Development of an On-Line Liquid Chromatography-Electrospray

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been ...Miss...
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Chem. Res. Toxicol. 2002, 15, 1302-1308

Development of an On-Line Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay to Quantitatively Determine 1,N2-Etheno-2′-deoxyguanosine in DNA Ana Paula M. Loureiro, Sabrina A. Marques, Camila C. M. Garcia, Paolo Di Mascio, and Marisa H. G. Medeiros* Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, Avenida Prof. Lineu Prestes, 748, CEP-05508-900, Sa˜ o Paulo, Sa˜ o Paulo, Brazil Received May 6, 2002

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N2etheno-2′-deoxyguanosine in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/108 dGuo) of the etheno adduct from approximately 350 µg of crude DNA hydrolysate. Using the newly developed technique, basal levels of 1,N2-etheno-2′-deoxyguanosine were determined in commercial calf thymus DNA (1.70 ( 0.09 adducts/107 dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 ( 0.4 adducts/107 dGuo), and in untreated female rat liver DNA (5.22 ( 1.37 adduct/107 dGuo). The mutagenicity of 1,N2-etheno-2′-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N2-etheno-2′-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.

Introduction Unsubstituted promutagenic etheno adducts (Figure 1), mainly 1,N6-etheno-2′-deoxyadenosine (dAdo)1 and 3,N4-etheno-2′-deoxycytidine (dCyd), have been detected as background DNA lesions in rodent and human tissues at levels varying from 2 adducts per 1010 to 2.8 adducts per 106 parent nucleosides (1-10). Background levels of N2,3-etheno-2′-deoxyguanosine (N2,3-dGuo) in DNA have also been detected in some studies (11-13). However, the formation of 1,N2-etheno-2′-deoxyguanosine (1,N2-dGuo) in DNA in vivo has not yet been demonstrated with the current methodology (14). Much interest in exocyclic etheno adducts resulted from the detection of increased levels of dAdo and dCyd in DNA from human, rat, and mice tissues in clinical situations associated with oxidative stress, such as metal storage diseases (15, 16), chronic infections, and inflammation (17). The levels of these adducts were also shown to be high in colon polyps of patients with familial adenomatous polyposis, who later develop cancer of the * To whom correspondence should be addressed. Fax: ++ (55) 11 30912186. Phone: ++ (55) 11 30912153. E-mail:[email protected]. 1 Abbreviations: 1,N2-dGuo, 1,N2-etheno-2′-deoxyguanosine; dAdo, 1,N6-etheno-2′-deoxyadenosine; N2,3-dGuo, N2,3-etheno-2′-deoxyguanosine; N2,3-Gua, N2,3-etheno-guanine; 1,N2-Gua, 1,N2-ethenoguanine; dCyd, 3,N4-etheno-2′-deoxycytidine; dGuo, 2′-deoxyguanosine; dThd, 2′-deoxythymidine; dCyd, 2′-deoxycytidine; dAdo, 2′deoxyadenosine; GC/ECNCI-HRMS, gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry; LC, liquid chromatography; ESI/MS, electrospray ionization mass spectrometry; HPLC/ESI/MS-MS, high-performance liquid chromatography/electrospray ionization tandem mass spectrometry; MRM, multiple reaction monitoring; DME, Dulbecco’s modified Eagle’s medium; HPLC, high-performance liquid chromatography; S/N, signalto-noise ratio.

Figure 1. Structures of the unsubstituted etheno-DNA adducts. dR, deoxyribose.

colon (18). In an interesting study, Nair and co-workers (19) reported a large (40-fold) increase in the levels of dAdo and dCyd in white blood cell DNA of women consuming diets rich in polyunsaturated fatty acids. In addition, a correlation between the accumulation of arachidonic acid metabolites and the formation of dAdo and dCyd during the tumor-promoting phase of the mouse skin two-stage model of carcinogenesis has also been observed (20). In vitro studies have demonstrated that incubation of nucleosides or DNA with lipid peroxidation products results in the formation of unsubstituted etheno adducts (21-28). Possible known endogenous sources of these adducts are oxidized R,β-unsaturated aldehydes, such as 4-hydroxynonenal, crotonaldehyde, acrolein, and 2,4decadienal epoxides (21, 22, 25-32). Interestingly, 1,N6ethenoadenine was also formed from the reaction of DNA with 2-phosphoglycolaldehyde, a model for the 3′-phos-

10.1021/tx025554p CCC: $22.00 © 2002 American Chemical Society Published on Web 10/02/2002

On-Line LC/MS-MS Detection of Ethenodeoxyguanosine

phoglycolaldehyde residue generated by oxidation at C3 of 2′-deoxyribose in DNA (33). Considering the fact that exocyclic DNA adducts today are perceived as potential new tools in the study of oxidative stress and cancer etiology and in the assessment of the efficacy of chemopreventive agents on DNA damage and cancer risk (1, 34-36), the development of sensitive and selective methods that allow their levels in tissue DNA to be determined with precision is necessary. Several methods, including a competitive immunoassay (37), the ultrasensitive, highly specific gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (GC/ECNCI-HRMS) (14), and high-performance liquid chromatography/ tandem mass spectrometry (HPLC/ESI/MS-MS) with offline reversed-phase extraction to purify the adducts from milligram amounts of DNA (21, 38) have been employed to quantify DNA adducts. On-line immunoaffinity chromatography coupled with HPLC/ESI/MS-MS has recently been reported for the detection and automated quantification of trace levels of dCyd in crude DNA hydrolysates (8). A similar methodology has allowed for the detection of dAdo (2). Despite the development of ultrasensitive detection methods, however, the in vivo formation of 1,N2-dGuo has yet to be demonstrated. This work describes the development and validation of a sensitive method to quantitatively determine 1,N2dGuo from in vitro and in vivo samples, using on-line reversed-phase HPLC separation with tandem mass spectrometry detection by multiple reaction monitoring (MRM).

Experimental Section Chemicals. All the chemicals employed here were of the highest purity grade commercially available. 2′-Deoxyguanosine (dGuo), formic acid, sodium hydroxide, potassium phosphate, magnesium sulfate, saccharose, magnesium chloride, and 2-chloroacetaldehyde were acquired from Merck (Darmstadt, Germany). [15N5]-2′-Deoxyguanosine was provided by Cambridge Isotope Laboratories (Andover, MA). Chromatography grade acetonitrile was obtained from EM Science (Gibbstown, NJ). Chloroform and ethanol were supplied by Cine´tica Quı´mica (Sa˜o Paulo, Brazil). Fetal calf serum was acquired from Cultilab (Campinas, SP, Brazil) and H2O2 from Fluka Chemika (Buchs, Switzerland). All the other chemicals used were from Sigma (St. Louis, MO). Water was purified in a Milli-Q system (Millipore, Bedford, MA). DNA Modified in Vivo. Eleven-week-old untreated female Wistar rats were anesthetized by ether inhalation and immediately sacrificed. Their livers were removed, snap-frozen in liquid nitrogen, and stored at -80°C. CV1-P Cell Culture. Green monkey kidney fibroblasts (line CV1-P), kindly provided by Dr. Roge´rio Meneghini from the Center of Structural Molecular Biology, Laborato´rio Nacional de Luz Sı´ncroton, Campinas, Brazil, were routinely grown in Dulbecco’s modified Eagle’s medium (DME) pH 7.4, supplemented with 10% (v/v) fetal calf serum, 0.04 g of penicillin/L, and 0.094 g of streptomycin sulfate/L. The cells were incubated in a CO2/air atmosphere (1:19) at 37 °C and were allowed to grow until they reached the confluence. DNA was extracted, hydrolyzed, and analyzed as described below. DNA Extraction. DNA was isolated by the chaotropic NaI method, as previously described (39, 40). Briefly, the tissues (1000 mg) or the cellular pellets (3 × 108 cells) were homogenized in 10 mL of a lysis solution [320 mM sucrose, 5 mM MgCl2, 10 mM Tris-HCl, 0.1 mM desferroxamine, and 1% (v/v) Triton X 100, pH 7.5]. After centrifugation at 1500g for 10 min,

Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1303 the pellets were suspended in 10 mL of the lysis solution and centrifuged at 1500g for 10 min. The pellets were then suspended in 6 mL of 10 mM Tris-HCl buffer, pH 8.0, containing 5 mM EDTA, 0.15 mM desferroxamine, and 350 µL of SDS 10%. The RNase A (30 µL, 10 mg/mL) and RNase T1 (4 µL, 20 units/ µL) in 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 2.5 mM desferroxamine, were added, and the reaction mixture was incubated at 37 °C. After 1 h, 300 µL of proteinase K (20 mg/mL) were added, followed by an additional incubation at 37 °C for 1 h. After centrifugation at 5000g for 15 min, the liquid phase was collected and 4 mL of a solution containing 7.6 M NaI, 40 mM Tris-HCl (pH 8), 20 mM EDTA, and 0.3 mM desferroxamine were added, followed by the addition of 8 mL of 2-propanol. The content in the tube was well mixed by inversion until a whitish precipitate appeared. The precipitate was collected by centrifugation at 5000g for 15 min and washed with 5 mL of 2-propanol 60% (5000g, 15 min), followed by 5 mL of ethanol 70% (5000g, 15 min). The DNA pellet was solubilized in 500 µL of desferroxamine (0.1 mM). The DNA concentration was measured spectrophotometrically at 260 nm. Enzymatic Hydrolysis of DNA. A total of 4 µL of 1 M sodium acetate buffer (pH 5) and 2 pmol of [15N5]-1,N2-dGuo were added to an aliquot of 0.1 mM desferroxamine solution containing 200 µg of DNA, followed by digestion with 2 units of nuclease P1 at 37 °C for 30 min. A total of 12 µL of 1 M TrisHCl buffer (pH 7.4), 12 µL of phosphatase buffer, and 6 units of alkaline phosphatase were then added for an additional 1 h incubation at 37 °C. The final volume of the solution was adjusted to 200 µL with water. The enzymes were precipitated by the addition of one volume of chloroform and, after centrifugation at 1000g for 5 min, the resulting aqueous layer was subjected to HPLC/ESI/MS-MS analysis (100 µL of the DNA solution/injection). The amounts of the reagents and labeled internal standard were proportionally adjusted for hydrolysis and analysis of other DNA quantities. 2′-Deoxyguanosine Quantification in DNA Samples. The following HPLC system was used to quantitatively determine dGuo in the DNA hydrolysates: a Luna 10 C18 (2) (250 mm × 10 mm i.d., 10 µm) semipreparative column (Phenomenex, Torrance, CA) was eluted with a gradient of water and acetonitrile (from 0 to 5 min, 5% acetonitrile; from 5 to 30 min, 5 to 20% acetonitrile) at a flow rate of 4.7 mL/min, and the absorbance was monitored at 254 nm. A standard calibration curve prepared within the range of dGuo expected to be present in the DNA hydrolysates was used for this quantification. Synthesis of 1,N2-Etheno-2′-deoxyguanosine Unlabeled Standard. The 1,N2-dGuo unlabeled standard was obtained by reacting dGuo with chloroacetaldehyde, as described by Loureiro et al. (21). Its identity was confirmed by the following spectroscopic features: UV λmax 222 ( ) 40 570 M-1 cm-1), 271 ( ) 10 971 M-1 cm-1), 295 nm ( ) 11 912 M-1 cm-1) at pH 1 (50 mM HCl-KCl); 226 ( ) 49 937 M-1 cm-1), 285 nm ( ) 16 785 M-1 cm-1) at pH 7 (50 mM phosphate buffer); and 233 ( ) 42 764 M-1 cm-1), 280 ( ) 8643 M-1 cm-1), 307 nm ( ) 11 687 M-1 cm-1) at pH 11 (50 mM carbonate-bicarbonate buffer). ESI/MS m/z 176 ([M + H]+ - 2-D-erythro-pentose, 65% relative intensity), 292 ([M + H]+ , 100% relative intensity). 1H NMR (DMSO-d6) δ 7.97 (s, 1H, H-2), 7.49 (d, 1H, J ) 2.43 Hz, H-6), 7.24 (d, 1H, J ) 2.58 Hz, H-7), 6.22-6.25 (dd, 1H, H-1′), 5.32 (bs, 1H, OH-5′), 5.23 (s, 1H, OH-3′), 4.37 (m, 1H, H-3′), 3.83-3.86 (m, 1H, H-4′), 3.58-3.60 (m, 1H, H-5′), 3.49-3.52 (m, 1H, H-5′′), 2.64-2.69 (m, 1H, H-2′), 2.17-2.21 (m, 1H, H-2′′). Synthesis of [15N5]-1,N2-Etheno-2′-deoxyguanosine Internal Standard. [15N5]-1,N2-dGuo was obtained by reacting [15N5]-dGuo with chloroacetaldehyde, with subsequent purification by HPLC, as described by Loureiro et al. (21). The identity of the [15N5]-adduct was confirmed by mass spectrometry analysis. ESI/MS for [15N5]-1,N2-dGuo: m/z 181 ([M + H]+ 2-D-erythro-pentose, 58% relative intensity), 297 ([M + H]+, 100% relative intensity). High-Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry (HPLC/ESI/

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MS-MS). On-line HPLC/ESI/MS-MS analyses in the positive mode were carried out using a Quattro II mass spectrometer (Micromass, Manchester, U.K.). The 1,N2-dGuo adduct in DNA samples was detected by multiple-reaction monitoring (MRM). A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of an autosampler (SIL-10AD/VP), an automated switching valve (FCV-12AH), two pumps (Class LC 10AD), an SPD-10AV/ VP UV detector controlled by a communication bus module (SCL-10A/VP-CBM 10A) and Class-VP software were used for sample injection and cleanup of the analytical column [Luna C18(2) 250 mm × 4.6 mm i.d., 5 µm, Phenomenex, Torrance, CA]. The adduct was eluted from this first column with a gradient of formic acid (0.1% in water) and acetonitrile (from 0 to 18 min, 8% acetonitrile and 0.4 mL/min; from 18 to 19 min, 8 to 50% acetonitrile and 0.4 to 0.2 mL/min; from 19 to 19.5 min, 50% acetonitrile and 0.2 to 0.1 mL/min; from 19.5 to 32 min, 50% acetonitrile and 0.1 mL/min; from 32 to 32.5 min, 50% acetonitrile and 0.1 to 0.05 mL/min; from 32.5 to 35 min, 50% acetonitrile and 0.05 mL/min; from 35 to 36 min, 50% acetonitrile and 0.05 to 0.8 mL/min; from 36 to 38 min, 50% acetonitrile and 0.8 mL/min; from 38 to 39 min, 50 to 8% acetonitrile and 0.8 mL/min; from 39 to 49 min, 8% acetonitrile and 0.8 to 0.4 mL/min). A third HPLC pump (LC-10AD, Shimadzu) was used to simultaneously load a second column [C18 (2) PhenosphereNext 150 mm × 2 mm i.d., 3 µm, Phenomenex] with an isocratic flow (0.05 mL/min) of a (50:50) solution of formic acid 0.1% in water:acetonitrile, maintaining a constant flow of the mobile phase to the mass spectrometer during the analysis. The position of the switching valve was changed twice: at 24 min, allowing the first column eluent to enter the second column; and at 35 min, permitting the first column to be washed while the adduct was eluted through the second column to the mass spectrometer. The total time spent on this analysis was 50 min. The DNA hydrolysates containing 1 pmol of the [15N5]-1,N2dGuo internal standard were injected into the above-described system. The m/z 292 f 176 (1,N2-dGuo) and 297 f 181 ([15N5]1,N2-dGuo) transitions were monitored with a dwell time of 1 s. The cone voltage was kept at 15 V. The collision energy was set at 10 eV. The pressure of the collision gas (argon) in the gas cell was adjusted to 6.7 × 10-4 mbar. The source temperature was held at 100 °C, and the drying and nebulizing gas (nitrogen) flow rates were optimized at 350 and 10 L/h, respectively. The capillary and HV electrode potentials were set at 3.10 and 0.30 kV, respectively. All the other parameters of the mass spectrometer were adjusted for acquisition of the best [M + H]+/[M + H]+ - 2-D-erythro-pentose transition. The data were processed using MassLynx software 3.2 (Micromass). For the acquisition of mass spectra, approximately 200 fmol of 1,N2-dGuo or [15N5]-1,N2-dGuo were injected into the abovedescribed system. Full scan data in MS1 and product ion mass spectra in MS2 were collected over a mass range 100-400 Da.

Results Method Development. In this paper, we report on the development of a new HPLC/ESI/MS-MS assay for the quantitative determination of 1,N2-dGuo in DNA. Scheme 1 outlines the entire assay procedure. The stable isotopic internal standard [15N5]-1,N2-dGuo, synthesized as described in the Experimental Section, was quantified with the UV extinction coefficient of the unlabeled 1,N2dGuo. The internal standard, 5 units of mass larger than the native analyte, was added to the DNA samples prior to enzymatic hydrolysis. The use of this internal standard ensures high specificity and accurate quantification of the 1,N2-dGuo adduct. The full scan and product ion mass spectra of 1,N2-dGuo and [15N5]-1,N2-dGuo are presented in Figure 2. The predominant ion in the full scan mass spectrum of 1,N2-dGuo is m/z 292, the [M + H]+ ion of the adduct (Figure 2A). The collision-induced

Loureiro et al. Scheme 1. HPLC/ESI/MS-MS Assay Procedure for 1,N2-EdGuo Detection in DNA

dissociation (CID) of the molecule under the conditions described in the Experimental Section revealed a predominant fragment at m/z 176, which is the [M + H]+ 2-D-erythro-pentose ion of the adduct (Figure 2B). A similar result was obtained for the isotopic [15N5]-1,N2dGuo standard (Figure 2, panels C and D), except that the corresponding ions were, as expected, 5 Da greater than those of the unlabeled adduct. The m/z transitions from 292 to 176 (1,N2-dGuo) and from 297 to 181 ([15N5]1,N2-dGuo) were therefore chosen for the MRM detection experiments. Figure 3 shows the reconstructed ion chromatograms obtained by MRM detection using the respective m/z transitions for 1,N2-dGuo and [15N5]-1,N2-dGuo. It is important to note that the presence of five atoms of 15 N in the internal standard of 1,N2-dGuo does not induce a detectable shift in its retention time with respect to that of the unlabeled adduct. The peak in the 1,N2dGuo standard chromatogram corresponds to the detection of 20 fmol of this adduct, with a signal-to-noise ratio (S/N) of 3:1. This was the limit of detection obtained for the standard injection. The need to isolate an analyte from the excess amount of normal DNA bases prior to injection into the mass spectrometer is one of the main challenges encountered in the use of HPLC/ESI/MS-MS for analyses of the low levels of DNA adducts present in biological samples. To overcome the problems that may arise from handling the samples during an off-line prepurification step (e.g., analyte loss, analyte contamination), we used a liquid chromatography method that allows the crude DNA hydrolysates to be directly analyzed for the presence of 1,N2-dGuo. The program developed for the liquid chromatography events (Scheme 2B) allowed for adequate separation of the adduct from the last eluted nucleoside, 2′-deoxythymidine (dThd), as shown in Figure 4. In addition to the solvent flow and the acetonitrile content, the use of 0.1% formic acid in the mobile phase was particularly important to allow for the elution of the normal nucleosides in the following order: dCyd < dAdo < dGuo < dThd. When pure water was used instead of 0.1% formic acid, the last eluted nucleoside was dAdo,

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Figure 2. Full scan (A, C) and product ion mass spectra (B, D) of 1,N2-dGuo and [15N5]-1,N2-dGuo, respectively. The labeled nitrogen atoms in the molecule are indicated with an asterisk. Conditions were as described in the Experimental Section.

Figure 3. Detection of 20 fmol of the 1,N2-dGuo standard by HPLC/ESI/MS-MS analysis in the MRM mode. (A) 1,N2dGuo: m/z 292 f 176; (B) [15N5]-1,N2-dGuo: m/z 297 f 181. Conditions were as described in the Experimental Section.

whose tailing was frequently a problem that reduced the sensitivity of the mass spectrometer for adduct detection. The automated switching valve (Scheme 2A), programmed to change its position in the interval between the end of elution of dThd and the beginning of elution of 1,N2-dGuo + [15N5]-1,N2-dGuo, ensured that no excess of normal nucleosides entered the mass spectrometer, preventing the loss of its sensitivity. Elution of the adduct through the second narrow bore column at a low flow rate allowed for further purification of the analyte after possible interference, as well as the adduct’s concentration into a straight peak, increasing the sensitivity of detection. Assay Calibration. The linearity and precision of the method over the range of 1,N2-dGuo content expected in the DNA samples were demonstrated by the calibration curve shown in Figure 5 (r2 ) 0.999, standard

deviation ) 6.7%). The curve was constructed from 10 different concentration points by plotting the chromatographic peak area ratios of 1,N2-dGuo to [15N5]-1,N2dGuo in each injection versus the amount (femtomoles) of the unlabeled adduct injected (5-5000 fmol/injection). The amount of injected 15N5-labeled internal standard was kept constant at 1000 fmol. Blank injections (sample solvent plus 1000 fmol of [15N5]-1,N2-dGuo) in the same conditions revealed that no carryover of the native 1,N2dGuo from previous analyses occurred. These injections also revealed that the 15N5-labeled internal standard was uncontaminated with any detectable level of the unlabeled adduct (data not shown). Method Validation and Application. The limit of detection for 1,N2-dGuo present in DNA samples was inferred from the analysis of untreated calf thymus DNA. When triplicate samples containing 354 µg of the DNA hydrolysates were analyzed by the HPLC/ESI/MS-MS system, the 1,N2-dGuo value was 1.70 ( 0.09 adducts/ 107 dGuo (a total of 46.36 ( 1.18 fmol of the adduct). The reconstructed ion chromatogram of one of these samples is depicted in Figure 6, from which the S/N ) 7:1 for the peak in the m/z 292 f 176 transition chromatogram (unlabeled 1,N2-dGuo) was calculated. On the basis of this observation, we found that the method’s detection limit for 1,N2-dGuo (S/N ) 3:1), when an analysis of approximately 350 µg of DNA hydrolysate is made, is about 7.4 adducts/108 dGuo (a total of ∼20 fmol of the adduct). Had larger amounts of DNA been used, this limit would have decreased. Analyses of different amounts of hydrolyzed calf thymus DNA (20, 50, 75, 100 µg) contaminated with 100 to 1000 fmol of 1,N2-dGuo standard prior to sample hydrolysis revealed that larger DNA samples do not cause suppression of the adduct’s response. The relative standard deviations (RSD) for the

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Loureiro et al.

Scheme 2. (A) Positions of the Automated Switching Valve during the HPLC/ESI/MS-MS Analysis. (B) Liquid Chromatography Program for the HPLC/ESI/MS-MS Analysis of 1,N2-EdGuoa

a

Mobile phase content: 0.1% of aqueous formic acid with the indicated % of acetonitrile (ACN).

Figure 4. Elution profile of a mixture of normal nucleosides (corresponding to approximately 50 µg DNA) with 1,N2-dGuo standard (500 pmol) through the first analytical column of the on-line HPLC/ESI/MS-MS system. The absorbance was monitored at 280 nm. Conditions were as described in the Experimental Section.

Figure 5. HPLC/ESI/MS-MS calibration curve for 1,N2-etheno2′-deoxyguanosine obtained by plotting the relative area ratios of 1,N2-dGuo (m/z 292) to the isotopically 15N5-labeled adduct (m/z 297) vs increasing amounts of 1,N2-dGuo. Conditions were as described in the Experimental Section.

adduct signal in the different amounts of DNA contaminated with 100, 300, 500, and 1000 fmol of 1,N2-dGuo were, respectively, 16.54, 3.88, 1.26, and 4.00%.

Figure 6. Detection of 1,N2-dGuo in 354 µg of untreated calf thymus DNA by HPLC/ESI/MS-MS analysis in the MRM mode. (A) 1,N2-dGuo: m/z 292 f 176. (B) [15N5]-1,N2-dGuo: m/z 297 f 181. Conditions were as described in the Experimental Section.

The accuracy of the method was evaluated by spiking 10-500 fmol of the unlabeled 1,N2-dGuo standard with 61 µg of untreated female rat liver DNA. All the standards were added prior to DNA hydrolysis. The basal level of 1,N2-dGuo encountered in the liver DNA sample (61 µg) prior to contamination with the adduct standard was 9.43 ( 0.87 adducts/107 dGuo (43.68 ( 4.03 fmol of 1,N2-dGuo/DNA sample, triplicate analyses). With the addition of 10, 50, 100, and 500 fmol of the 1,N2-dGuo standard, the levels of 1,N2-dGuo detected in the DNA samples rose, respectively, to 52.37 ( 6.60, 96.16 ( 4.19, 147.72 ( 6.93, and 563.14 ( 14.14 fmol. These values accurately reflected the known additional amounts of adduct. Analyses of liver DNA samples (130 µg) from five other untreated female Wistar rats confirmed the occurrence of a natural rat liver basal level of 5.22 ( 1.37 1,N2dGuo/107 dGuo. The reconstructed ion chromatogram of

On-Line LC/MS-MS Detection of Ethenodeoxyguanosine

Figure 7. HPLC/ESI/MS-MS detection of 1,N2-dGuo in 130 µg of DNA extracted from the liver of an untreated female Wistar rat. MRM mode was used. (A) 1,N2-dGuo: m/z 292 f 176; (B) [15N5]-1,N2-dGuo: m/z 297 f 181. Conditions were as described in the Experimental Section.

one of these samples is shown in Figure 7. This is the first time that the existence of a basal level of the adduct 1,N2-dGuo is demonstrated in vivo. The HPLC/ESI/MS-MS method developed here was also applied in an investigation of the occurrence of a basal level of 1,N2-dGuo in cultured CV1-P cells. The method’s precision was confirmed when five cell DNA samples (124 µg) were analyzed on two different occasions. On the first occasion, the 1,N2-dGuo level was found to be 4.48 ( 0.42 adducts/107 dGuo (n ) 5). On the second occasion, the measured level was 4.55 ( 0.37 adducts/107 dGuo (n ) 5).

Discussion A method to quantitatively determine 1,N2-dGuo in DNA samples was developed, using on-line HPLC separation with tandem mass spectrometry detection by multiple reaction monitoring (MRM). This innovative method improves the analytical assay previously reported for 1,N2-dGuo quantification in DNA hydrolysates (21). The major advantage of this method is the on-line adduct separation from normal nucleosides coupled with the accuracy provided by MS-MS detection. The method solves the problems involved in off-line prepurification steps, allowing for the direct analysis of crude DNA hydrolysates. The addition of an isotopically labeled internal standard prior to DNA hydrolysis improves the method’s confidence level since it allows for the correction of any possible loss of analyte during the procedure. This newly developed methodology enabled us to determine basal levels of 1,N2-dGuo in commercial calf thymus DNA (1.70 ( 0.09 adducts/107 dGuo), in cultured CV1-P cells DNA (4.5 ( 0.4 adducts/107 dGuo), and in female rat liver DNA (5.22 ( 1.37 adducts/107 dGuo). Morinello and co-workers (14) recently used the immunoaffinity/GC/ECNCI-HRMS technique to quantify basal levels of N2,3-Gua in commercial calf thymus DNA, which was found to be 0.8 adducts/107 Gua. Although the IA/GC/ECNCI-HRMS was as sensitive as the method described here for the quantification of 1,N2-dGuo, the presence of 1,N2-dGuo as an endogenous DNA lesion could not be demonstrated (14). This, therefore, is the first report of the in vivo detection and quantification of 1,N2-dGuo as a DNA

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basal lesion. The basal levels found here are comparable with those of dCyd (11 adducts/108 normal nucleotides or ∼4.4 adducts/107 dCyd) encountered in rat liver DNA using a similar methodology (8). Higher amounts of endogenous dAdo (25 adducts/107 dAdo) were detected in human placental DNA, using three different methodologies (GC/MS, LC/MS, and HPLC/fluorescence), as reported by Chen and co-workers (3). The biological relevance of the occurrence of 1,N2dGuo in DNA has been revealed by studies showing the mutagenicity of this etheno adduct. In vitro misincorporation studies found that 1,N2-dGuo tends to strongly block replication at and beyond the site of substitution and misincorporation of dATP and dGTP across it was also observed (41). Miscoding opposite this adduct in an Escherichia coli/M13MB19-based system was examined and it was shown to be mutagenic, with more abundant GfA mutations followed by GfT mutations (42). 1,N2dGuo was also shown to generate deletions, rearrangements, double mutants, and base pair substitutions near the 1,N2-dGuo site in mammalian cells DNA using a system to evaluate adduct intra-chromosomal site-specific mutagenesis (43). Recently, Saparbaev et al. (44) described that 1,N2-etheno-guanine (1,N2-Gua) is a primary substrate of E. coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase. These data reinforce the potential biological relevance of this adduct. The possible use of etheno adducts as in vivo markers of DNA damage requires simple methodologies with high levels of confidence. The methodology described herein provides a highly precise and specific way to quantify the 1,N2-dGuo adduct in tissue DNA, allowing for the future evaluation of its levels when tissues are subjected to factors that promote or decrease the lipid peroxidation process.

Acknowledgment. This work was supported by the Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (Brazil), the Conselho Nacional para o Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq (Brazil), Pro´Reitoria de Pesquisa da Universidade de Sa˜o Paulo (Brazil), and Programa de Apoio aos Nu´cleos de Exceleˆncia, PRONEX (Brazil).

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