High-Resolution Mass

Patricia B. Upton,‡ Francis Johnson,| and James A. Swenberg*,†,‡,§. Departments of Pathology and of Environmental Sciences and Engineering and ...
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Immunoaffinity/Gas Chromatography/High-Resolution Mass Spectrometry Method for the Detection of N2,3-Ethenoguanine Amy-Joan L. Ham,† Asoka Ranasinghe,‡ Eric J. Morinello,‡,§ Jun Nakamura,‡ Patricia B. Upton,‡ Francis Johnson,| and James A. Swenberg*,†,‡,§ Departments of Pathology and of Environmental Sciences and Engineering and Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599, and Department of Phamacological Sciences, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794 Received August 13, 1999

Etheno adducts are formed after exposure to a number of carcinogens, including vinyl chloride, as well as endogenously as a result of lipid peroxidation. A sensitive and selective assay for N2,3-ethenoguanine (Gua) was developed using immunoaffinity (IA) columns made with polyclonal antibodies to Gua followed by gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry (GC/ECNCI/HRMS) analysis of its pentafluorobenzyl derivative. These IA columns were specific for Gua and did not bind guanine, deoxyguanosine, 1,N6-ethenoadenine, or 1,N2-ethenoguanine. The level of recovery of standards from the IA columns was 107 ( 7% and throughout the entire method (using nucleoside enzymatic digestion) with or without DNA was 72 ( 6%. Four different hydrolysis/digestion procedures were compared, nucleoside enzymatic (EZ), neutral thermal hydrolysis (NT), formic acid hydrolysis (FA), and HCl hydrolysis. All hydrolysis methods with subsequent IA chromatography produced linear standard curves with r2 values of 0.999 or better. The level of Gua in chloroethylene oxide-treated calf thymus DNA (CEO-ctDNA) was 38 ( 2, 42 ( 3, and 49 ( 2 fmol of Gua/µg of DNA using EZ, NT, and FA, respectively. These numbers remained consistent when the amount of DNA processed was doubled or tripled. These numbers were comparable to the previously published value of 55 ( 8 fmol of Gua/µg of DNA for the same DNA using HCl hydrolysis, cation exchange cleanup, and LC/MS analysis [Yen, T. Y., et al. (1996) J. Mass Spectrom. 31, 1271-1276]. Additionally, HCl hydrolysis of rat liver DNA from control and vinyl fluoride-exposed rats gave similar Gua results when compared to those from enzymatic digestion using this method. This method gave a detection limit of 5 Gua adducts/108 normal dGuo nucleosides in 150 µg of DNA using EZ and somewhat lower detection limits using NT and HCl hydrolysis. The method is more sensitive and selective than previously used methods for the quantitation of this adduct.

Introduction The DNA adduct N2,3-ethenoguanine (Gua)1 is formed from a number of carcinogens, including vinyl chloride (VC) (1). The formation of this adduct occurs from the reaction of guanine with electrophiles that are produced * To whom reprint requests should be sent: Department of Environmental Sciences and Engineering, CB #7400, University of North Carolina, Chapel Hill, NC 27599-7400. Telephone: (919) 966-6139. Fax: (919) 966-6123. † Department of Pathology, University of North Carolina. ‡ Department of Environmental Sciences and Engineering, University of North Carolina. § Curriculum in Toxicology, University of North Carolina. | State University of New York at Stony Brook. 1 Abbreviations: Gua, N2,3-ethenoguanine; Guo, N2,3-ethenoguanosine; dGuo, N2,3-ethenodeoxyguanosine; dGuo, deoxyguanosine; IA, immunoaffinity; GC/ECNCI/HRMS, gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry; EZ, nucleoside enzymatic digestion; NT, neutral thermal hydrolysis; FA, formic acid hydrolysis; CEO, chloroethylene oxide; CEO-ctDNA, chloroethylene oxide-treated calf thymus DNA; LC/MS, liquid chromatography/mass spectrometry; VC, vinyl chloride; Ade, 1,N6ethenoadenine; Cyt, 3,N4-ethenocytosine; KLH, keyhole limpet hemocyanin; PBS/azide, phosphate-buffered saline [10 mM phosphate buffer and 0.85% saline (pH 7.5)] with 0.2% sodium azide; SIM, selected ion monitoring; PFB, pentafluorobenzyl; PFK, perfluorokerosene.

from carcinogens as well as from endogenous sources, presumably from products of lipid peroxidation. In the case of VC, the highly reactive electrophile chloroethylene oxide (CEO), which is formed from the metabolic activation of vinyl chloride by cytochrome P450, alkylates DNA and forms a variety of etheno adducts, including Gua (2). The major VC-induced adduct, 7-(2′-oxoethyl)guanine, does not induce mutations in vitro (3), and thus is not thought to play a significant role in VC-induced mutagenesis (see Figure 1 for structures). However, the exocyclic derivatives 1,N6-ethenoadenine, 3,N4-ethenocytosine, N2,3-ethenoguanine, and 1,N2-ethenoguanine (Ade, Cyt, Gua, and 1,N2-Gua, respectively) have demonstrated miscoding potential in vitro and in vivo (410). Both in vitro and in vivo studies showed that Ade caused A to C transversions, Cyt caused C to A transversions and C to T transitions, and Gua caused G to A transitions. The results of these studies were consistent with the mutations observed in tumors from vinyl chloride-exposed humans and rats in two separate studies. In five out of six human liver angiosarcomas associated with vinyl chloride exposure, GC to AT transitions

10.1021/tx990150r CCC: $18.00 © 1999 American Chemical Society Published on Web 11/19/1999

IA/GC/MS Method for N2,3-Ethenoguanine

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and selectivity of the previously used assay, which is necessary for the study of endogenously formed Gua.

Experimental Procedures

Figure 1. Structures of DNA adducts formed from vinyl chloride exposure.

were observed at codon 13 of the c-Ki-ras-2 gene, consistent with either Gua or Cyt mutagenic properties (11). In another study, tumors from vinyl chloride-exposed rats exhibited G to A transitions, A to C transversions, and C to T transitions in N-ras genes, all of which are consistent with the promutagenic properties of etheno adducts (12). Although Gua is formed from VC exposure to a lesser extent than some other adducts, its greater mutagenic potential points to its possible significance in the carcinogenicity of a number of carcinogens. Fedtke et al. (13) originally reported that Gua appeared to have a very long half-life in hepatic DNA (exceeding 30 days). It is now believed, however, that in this original report the half-life of Gua could not be calculated accurately because baseline levels of endogenous Gua were being measured rather than the observed persistence of Gua after vinyl chloride treatment. The occurrence of DNA adducts from endogenous sources has become increasingly apparent. Most of these adducts have been postulated to occur from the reaction of DNA with products of lipid peroxidation or directly from reactive oxygen species. Chung and colleagues have shown that the related etheno adducts, 1,N2-ethenoguanine and 1,N6-ethenoadenine, are formed from the epoxide of the lipid peroxidation product trans-4-hydroxynonenal, 2,3-epoxy-4hydroxynonenal (14-17). Recently, we have also shown that Gua is formed as a result of the reaction between dGuo and 4-hydroxynonenal or ethyl linoleate under peroxidizing conditions (18). Since Gua may be formed from both endogenous and exogenous sources, it will be important to elucidate its formation from both sources, particularly with carcinogens that may produce Gua by both direct alkylation and increasing lipid peroxidation levels. To study these relationships and their relevance to carcinogenicity, a sensitive assay that can distinguish between endogenously and exogenously formed Gua is necessary. Previously, our laboratory developed a GC/ MS assay that utilized cation exchange chromatography for the initial cleanup of Gua, followed by GC/MS analysis of its pentafluorobenzyl derivative (19). In the study presented here, we have developed a GC/ECNCI/ HRMS method for Gua using enrichment by immunoaffinity chromatography cleanup. The necessity for a new assay originally arose since the cation exchange resin used previously was no longer commercially available, but also because of the need for a more sensitive and selective assay. This new assay improves the sensitivity

Materials. Spleen phosphodiesterase, alkaline phosphatase, and DNase I were purchased from Sigma Chemical Co. (St. Louis, MO). Snake venom phosphodiesterase was purchased from Worthington Biochemical (Freehold, NJ). Protein A Sepharose CL-4B was purchased from Pharmacia Biotech (Piscataway, NJ). Disposable polystyrene columns were purchased from Pierce (Rockford, IL). Pentafluorobenzyl bromide and acetone were obtained from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were obtained commercially and were of the highest purity available. Centricon 10 filters were obtained from Amicon, Inc. (Beverly, MA). Guo was synthesized by the method of Khazanchi et al. (20). Gua was synthesized by the method of Sattsangi et al. (21). Stable isotope internal standards of Gua were synthesized by first preparing isotopically labeled guanine which was synthesized by the method of Scheller et al. (22) with the modification that for [13C4,15N2]Gua, K13C15N and Na15NO2 were used to label N-7 and N-9. Isotopically labeled Gua was then synthesized by first protecting labeled Gua with a benzyl group at O6 using a two-step procedure involving acetylation at N2 and N9, according to the method of Zou and Robins (23), followed by Mitsunobu reaction, according to the method of Khazanchi et al. (20). The etheno bridge of Gua was then formed according to the method of Sattsangi et al. (21). Production of Polyclonal Antibodies. Conjugation of Guo to keyhole limpet hemocyanin (KLH) was achieved by a modification of the periodate oxidation procedure of Erlanger and Beiser (24). Guo was oxidized in 50 mM NaIO4 for 20 min at room temperature, and the reaction was stopped with the addition of 1 M ethylene glycol (3% v/v). The oxidized product was then reacted with KLH in 50 mM sodium borate buffer (pH 8.0) for 35 min at room temperature. After the addition of 0.25 volume of 0.8 M NaBH3, the mixture was incubated for 1 h at room temperature and maintained at pH 8.0 with the addition of 2 M Na2CO3. The pH was adjusted to 7.4 with 0.2 M HCl and incubated at room temperature for 15 min, followed by incubation at 4 °C for 2 h. The resulting conjugate was purified on a Sephadex G-50 column (1.5 cm × 30 cm) to ensure the separation of any uncoupled adduct from the conjugate. The quantity of protein and nucleosides was determined by UV absorbance at 280 and 260 nm, respectively. The purified solution was then lyophylized and stored desiccated at -70 °C. Conjugates were checked for stability by gel filtration at 2, 4, 8, and 16 weeks. Three New Zealand white rabbits (Charles River Laboratories, Raleigh, NC) were immunized with the Guo-KLH conjugate using “Procedure 2” from the methods of Muller and Rajewsky (25). Preparation of Immunoaffinity Columns. Polyclonal antiserum was coupled to the Protein A Sepharose gel using the method of Friesen et al. (26) with the modification that, to make four columns, 500 µL of antisera was added to 1 mL of washed Protein A Sepharose. Once the resin had been prepared, the resin was resuspended in 4 mL of phosphate-buffered saline [10 mM phosphate buffer and 0.85% saline (pH 7.5)] with 0.2% sodium azide (PBS/azide), and each column was prepared using 1 mL of resin. If more than four columns were being prepared at a given time, all resin was combined prior to preparation of the columns to increase the uniformity of the columns. Prior to use, the columns were washed at least three times with 10 mL of PBS/azide, 10 mL of water, 10 mL of methanol, 5 mL of water, 10 mL of 0.1 M formic acid, 10 mL of water, and 10 mL of PBS/ azide. HPLC. An HPLC system equipped with two Water Associates (Milford, MA) 510 pumps, either a 712 WISP autoinjector or a Rheodyne injector, an Applied Biosystems 757 UV detector, and/ or a Perkin-Elmer LS 40 fluorescence detector was used. The HPLC conditions were as follows. In system 1, a Whatman

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Partisil 10 SCX, 4.6 mm × 250 mm column was eluted isocratically with 25 mM ammonium formate and 10% methanol (pH 2.8) at a rate of 1.8 mL/min (for IA column characterization). System 2 was the same as system 1 except the mobile phase was 100 mM ammonium formate and 10% methanol (pH 2.8) (for Gua analysis). In system 3, a Beckman Ultrasphere ODS, 5 µm, 4.6 mm × 250 mm column was run isocratically with 50 mM potassium phosphate (monobasic) and 10% methanol (pH 5.5) (for dGuo analysis). Assay Procedures. Enzymatic Digestion. DNA (1.8-5.4 µg for CEO-ctDNA or 200-400 µg for rat liver DNA) was subjected to enzymatic digestion to the nucleoside by the method of Richter et al. (27) in the presence of 200 or 75 fmol of [13C4,15N2]-N2,3-Gua or [13C4]-N2,3-Gua, as internal standards. Following digestion of the DNA, the enzymes were removed from the resulting deoxynucleosides by Centricon 10 filtration (5000g for 1 h at 4 °C) using a Sorvall RC5C centrifuge (Sorvall Products, L. P., Newtown, CT). An aliquot was then taken for dGuo analysis, and the samples were adjusted to neutral pH with an equal volume of 40 mM Tris-HCl buffer (pH 6.0). Samples were then purified by IA chromatography as described below. Following IA purification, the samples were hydrolyzed to the corresponding nucleobase in 200 µL of 1% formic acid at 60 °C for 45-60 min and then dried under vacuum using a Speed Vac (Savant, Holbrook, NY). Samples were then prepared for GC/ECNCI/MS analysis as described below. Neutral Thermal Hydrolysis. DNA was hydrolyzed by neutral thermal hydrolysis by incubating samples in 500 µL of 50 mM sodium phosphate buffer (pH 7.4) at 100 °C for 20 min and then immediately cooling on ice. Samples were then separated from the DNA backbone by Centricon 10 filtration (5000g for 1 h at 4 °C), including a 0.5 mL water wash of the filter for quantitative transfer of the hydrolyzed Gua base. The DNA backbone was retrieved by inversion of the filter and centrifugation at 1000g for 2 min, according to the manufacturer’s instructions. The filter was inverted again and washed with 0.5 mL of water, and any residual DNA was retrieved from the filter, again by inversion of the filter and centrifugation at 1000g for 2 min. The resulting DNA was then subjected to HCl hydrolysis (0.1 M HCl in 1 mL at 70 °C for 30 min) for guanine analysis. Gua samples were then processed by IA chromatography and prepared for GC/ECNCI/MS analysis as described below. HCl Hydrolysis. DNA was hydrolyzed by HCl hydrolysis in 0.5 mL of 0.1 M HCl at 70 °C for 30 min. Following hydrolysis, nucleobases were separated from the DNA backbone by Centricon 10 filtration (5000g for 1 h at 4 °C), and a 100 µL aliquot was removed for guanine analysis. The filters were then rinsed with 2 volumes of PBS/azide and 0.09 volume of 1 N sodium hydroxide for quantitative transfer of the hydrolyzed Gua base and so a sample with neutral pH could be obtained. Samples were purified by IA chromatography and derivatized for GC/ ECNI/MS analysis as described below. Formic Acid Hydrolysis. DNA was hydrolyzed by formic acid hydrolysis by resuspending a dried DNA sample in 200 µL of 1% formic acid and incubated at 60 °C for 45 min. Samples were then dried under vacuum and resuspended in 1 mL of PBS. Following hydrolysis, nucleobases were separated from the DNA backbone by Centricon 10 filtration (5000g for 1 h at 4 °C), and a 100 µL aliquot was removed for guanine analysis. Samples were purified by IA chromatography and derivatized for GC/ ECNI/MS analysis as described below. Immunoaffinity Purification. Samples were purified by immunoaffinity chromatography utilizing columns made by coupling polyclonal antibodies to Gua to the Protein A Sepharose CL-4B gel as described above. Samples were loaded on IA columns with 2 mL of PBS/azide. Immediately following sample loading on the column, the columns were washed with 5 mL of PBS/azide, 5 mL of water, and 10 mL of 10% methanol. Samples were then eluted with 5 mL of 100% methanol into silanized glass tubes and dried under vacuum using a Speed Vac.

Ham et al. Columns were regenerated by washing with 5 mL of methanol, 5 mL of water, 10 mL of 0.1 M formic acid, 10 mL of water, and 10 mL of PBS/azide. All procedures were performed at 4 °C except for sample loading and sample elution which were performed at room temperature. IA columns were stored at 4 °C in PBS/azide. The level of recovery of Gua from the IA columns was checked periodically by applying 200 fmol of Gua (but no internal standard) to the columns and collecting the eluant in a tube containing 75 fmol of internal standard. The amount of Gua recovered was calculated on the basis of a standard curve. Columns were stable at 4 °C and did not show any loss of recovery level for well over 10 uses over a period of 2 months. Derivatization of EGua for GC/ECNCI/MS Analysis. Following IA cleanup (and formic acid hydrolysis in the case of enzymatic digestion), samples were derivatized to their corresponding pentafluorobenzyl derivatives according to the method of Fedtke et al. (19) with minor modifications. Briefly, 25 mg of oven-dried (70 °C) potassium carbonate, 500 µL of acetone, and 35 µL of 5% pentafluorobenzyl bromide in acetone were added to the dried sample. Samples were then incubated at 50 °C for 70-75 min while being mixed constantly using a Mistral mixer (Lab-Line Instruments, Melrose Park, IL) and then dried under nitrogen at 45 °C. Samples were cleaned up by solid-phase extraction by dissolving the residue in 200 µL of dichloromethane, applying to silica gel columns (prepared via the method described in ref 19), washing with 4 mL of hexane and 6 mL of 5% ethyl acetate in hexane, and eluting in 3 mL of ethyl acetate. Samples were then dried under nitrogen and redissolved in 15-50 µL of toluene for analysis by GC/ECNCI/HRMS. Gas Chromatography/High-Resolution Mass Spectrometry of Pentafluorobenzyl Derivatives. The analyses were performed on a Hewlett-Packard 5890 GC interfaced to a VG70250SEQ mass spectrometer. A DB-5MS fused silica capillary column (30 m × 0.32 mm, 0.1 µm thickness; J & W Scientific) with a helium (99.999% pure, Sunox) head pressure of 10 psi was used in all experiments. The samples were introduced using the direct injection mode utilizing Restek Uniliner model 20355. The injection port temperature was 290 °C, and the initial column temperature was 100 °C for 30 s, increasing at a rate of 25 °C/min to 150 °C and at a rate of 15 °C/min to 300 °C and held for 3 min. Selected ion monitoring (SIM) experiments at a mass resolving power of 10 000 were performed by monitoring the fragment ions, (M - PFB)- [m/z 354.0413 (Gua), 358.0549 (13C4-labeled internal standard), 360.0489 (13C4- and 15N2-labeled internal standard)]. The perfluorokerosene (PFK) lock mass and the dwell time were 354.9792 amu and 50 ms/ion, respectively. The methane (99.99% pure, Airco) pressure was adjusted to give the ion gauge reading of 3 × 10-5 mbar. The CI slit in the EI/ CI source was selected, and the instrument was tuned using the PFK mass (m/z 293) under the CI negative ion mode. The electron energy and emission current were also optimized such that highest ion abundance of m/z 293 from PFK (PCR Inc.) was displayed on the oscilloscope. After the tuning, the electron energy and emission current ranged between 60 and 100 eV and between 0.5 and 1.0 mA, respectively.

Results and Discussion In this study, we report the development of a new IA/ GC/ECNCI/HRMS assay for the detection of Gua. Scheme 1 shows a diagram depicting the overall assay procedures. This assay has increased the sensitivity and selectivity over those of the previously used assay for Gua through the use of polyclonal antibodies to enrich Gua from DNA samples. In this study, we have also investigated the use of several different methods for hydrolysis/digestion of DNA to determine the optimal conditions for the detection of this adduct using immunoaffinity as a cleanup method and to confirm the accuracy of the method.

IA/GC/MS Method for N2,3-Ethenoguanine Scheme 1. IA/GC/NICI/HRMS Assay Procedures

Polyclonal antibodies were raised against Gua using N2,3-ethenoguanosine (Guo) conjugated with KLH. Immunoaffinity columns, prepared with the resulting antisera, were characterized for their affinity for Gua, dGuo, Ade, 1,N2-Gua, and Gua. To determine the separation of compounds on the IA columns, Gua, dGuo, Ade, 1,N2Gua, and Gua were loaded on IA columns, and fractions were collected and analyzed by either HPLC (Gua, dGuo, Ade, and Gua) or GC/MS (Gua and 1,N2-Gua, using the same derivatization procedure that was used for Gua). This method clearly separates Gua from Gua,

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since all of the Gua is eluted in the loading fraction and Gua is retained on the columns until elution with methanol. A similar profile was seen when dGuo was loaded on the IA columns. In addition, Ade and 1,N2Gua were also separated from Gua using IA columns since neither of these was detected in the elution fraction. These results demonstrate the clear separation from a number of compounds that could interfere with analysis of Gua by this method. Table 1 demonstrates the high level of recovery of Gua from the IA columns. There was a decrease in the level of recovery when samples were processed through the entire method (using enzymatic digestion procedures) as compared to the level of recovery of standards loaded directly onto the columns (72 ( 6 vs 107 ( 7%, respectively). This decrease can be ascribed to the loss of sample during sample transfer, Centricon filtration, etc. The level of recovery of Gua was the same (72%) for standards run through the entire method in the presence or absence of DNA. Additionally, no loss in recovery level was seen when 400 pmol of Gua was loaded directly onto the columns, indicating that the columns had a binding capacity for Gua of greater than 400 pmol. The high level of recovery, along with complete separation from Gua, dGuo, and 1,N2-Gua, has allowed for an improvement in selectivity over the previously used cation exchange cleanup method for Gua (19). This is clearly demonstrated in Figure 2 in which a CEO-treated rat liver sample was first processed by cation exchange using the method of Fedtke et al. (19). The sample was then split and either directly analyzed by LC/MS using the method of Yen et al. (28) or processed using IA cleanup followed by LC/MS analysis. It is clear, in Figure 2, the IA cleanup not only gave a more pure sample (as demonstrated by the disappearance of other peaks that were present) but also decreased the background in the sample. This method, therefore, increased the selectivity of the method over that of the previously used cation exchange cleanup. Representative chromatograms of endogenous Gua in rat liver samples using enzymatic digestion, neutral thermal hydrolysis, and HCl hydrolysis are shown in Figure 3. This figure clearly demonstrates the ability to detect endogenous levels of Gua in biological samples.

Figure 2. LC/MS analysis of HCl-hydrolyzed CEO-treated rat liver DNA following (A) cation exchange cleanup as described by Fedtke et al. (19) or (B) cation exchange cleanup followed by immunoaffinity chromatography.

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Ham et al.

Table 1. Levels of Recovery of Standards from Immunoaffinity Columns sample

% recovery

IA columnsa enzymatic digestion and IA columns (standards or rat liver DNA)b

107 ( 7 72 ( 6

a The level of recovery was determined by adding [13C ]Gua 4 after elution of Gua from the IA columns and comparing to standards not run over IA columns. Numbers are expressed as means ( SD (N ) 19). b The level of recovery was determined by adding [13C4,15N2]Gua after IA column elution of rat liver DNA samples or standards containing the [13C4]Gua internal standard and processed following enzymatic digestion procedures (see Experimental Procedures). The level of recovery was determined from the ratio of [13C4,15N2]Gua:[13C4]Gua and comparing this ratio to those of standards not run over IA columns. Numbers are expressed as means ( SD (N ) 17).

Figure 3. Representative GC/HRMS chromatograms from endogenous rat liver DNA samples following immunoaffinity cleanup. (A) Enzymatic digestion, 0.089 Gua adducts/106 normal dGuo nucleosides from ∼100 µg of DNA. (B) Neutral thermal hydrolysis, 0.086 Gua adducts/106 normal Gua bases from ∼100 µg of DNA. (C) HCl hydrolysis, 0.069 Gua adducts/ 106 normal Gua bases from ∼160 µg of DNA.

The limit of quantitation in DNA samples for this method using enzymatic digestion was approximately 5 fmol in the entire sample (500 amol injected onto the GC/MS system). This value for a biological sample is roughly equivalent to 5 Gua adducts/108 normal dGuo nucleosides in 150 µg of DNA and lower if larger amounts of DNA were used. Limits of quantitation were even lower using neutral thermal and HCl hydrolyses. Although the limit of detection previously reported in liver DNA

Figure 4. Representative standard curves from IA columns following enzymatic digestion (9, r2 ) 0.9996), neutral thermal hydrolysis (b, r2 ) 0.9995), or HCl hydrolysis (2, r2 ) 0.9996). The X-axis reflects the total amount of G in the sample. The Y-axis reflects the ratio of peak areas for analyte to internal standard. All curves were linear out to the 1000 fmol range (data not shown).

samples was 6 Gua adducts/108 normal Gua bases (19), the amount of DNA required to produce this signal was 3-4 times greater than that in the current method and was not routinely achievable. The current method, therefore, is 3-4 times more sensitive. Additionally, the current method is much less labor intensive than the previous method and allows for the processing of a larger number of samples at a given time. The previous method took approximately 2 days from hydrolysis until the samples were ready for GC/ECNCI/HRMS, and only eight samples could be processed at a given time. Although the current method takes about the same amount of time for processing, the procedures are much less laborious, and 3-4 times as many samples can be processed at the same time. Another advantage to this method over the previous cation exchange method is that the columns can be reused. This method makes the routine quantitation of endogenous Gua much more feasible since much less DNA is required for analysis. IA chromatography, using all of the hydrolysis/digestion methods described in this paper, produced a linear response of standards from 5 fmol to more than 1000 fmol (Figure 4, data for 100-1000 fmol not shown). Standards run through the entire procedure for enzymatic digestion, neutral thermal hydrolysis, and HCl hydrolysis all gave standard curves with an r2 of more than 0.999. Generally, if larger amounts of Gua were to be quantitated (in the 150-1000 fmol range), we used a larger amount of internal standard (200 vs 75 fmol). With greater amounts of Gua, the multiplier settings needed to be lowered to accurately detect the analyte. This lower multiplier setting might lead to the disappearance of the internal standard signal if the original amount of internal standard would be used. The amount of internal standard, therefore, was increased to ensure detection of both the analyte channel and internal standard channel without saturation of the detector. Figure 5 demonstrates the results of a comparison of hydrolysis/digestion methods using IA cleanup. Utilizing calf thymus DNA that was directly alkylated with CEO (CEO-ctDNA), both neutral thermal and formic acid hydrolyses, as well as enzymatic digestion, all gave similar numbers for the amount of Gua when normalized to the amount of DNA. When 1.8 µg of CEO-ctDNA was analyzed, these hydrolysis/digestion methods yielded amounts of 38 ( 2, 42 ( 3, and 49 ( 2 fmol of Gua/µg of DNA for enzymatic digestion, neutral thermal hydrolysis,

IA/GC/MS Method for N2,3-Ethenoguanine

Figure 5. Quantitation of Gua in CEO-treated calf thymus DNA using enzymatic digestion, neutral thermal hydrolysis, or formic acid hydrolysis. Numbers are expressed as means ( SD. Table 2. Comparison of DNA Digestion/Hydrolysis Methods of Rat Liver Using IA Cleanup for EGua Analysis Gua/106 Gua DNAa

control rat liver enzymatic digestion neutral thermal hydrolysis HCl hydrolysis vinyl fluoride-treated rat liver DNAb enzymatic digestion HCl hydrolysis

0.081 ( 0.009c 0.085 ( 0.010 0.073 ( 0.004 2.37 ( 0.03 2.44 ( 0.03

a For Sprague-Dawley rat liver, enzymatic hydrolysis and neutral thermal hydrolysis were carried out with the same DNA sample, and a separate sample was used for HCl hydrolysis. b DNA was from male Sprague-Dawley rats exposed to 2500 ppm vinyl fluoride for 6 h/day and 5 days/week for 4 weeks (31). c Numbers are expressed as means ( SD (N ) 7 for neutral thermal hydrolysis and N ) 3 for all other digestion/hydrolysis methods).

and formic acid hydrolysis, respectively. When the amount of CEO-ctDNA processed using these methods was doubled or tripled, these numbers remained consistent (34 ( 2 fmol of Gua/µg of DNA for enzymatic digestion using 3.6 µg of DNA or 40 ( 1 and 41 ( 1 fmol of Gua/ µg of DNA for neutral thermal hydrolysis using 3.6 and 5.4 µg of DNA, respectively). In addition, these numbers are comparable to the amount of Gua seen in this CEOctDNA when analyzed by cation exchange cleanup followed by LC/MS, which yielded a value of 55 ( 8 fmol of Gua/µg of DNA (28). On the basis of these results, it appears that both neutral thermal hydrolysis and enzymatic digestion give very comparable results. Formic acid hydrolysis also gave comparable values, but these numbers were consistently 20% higher than the values achieved with the other hydrolysis/digestion methods. We also demonstrated that enzymatic digestion of rat liver DNA from control and vinyl fluoride-treated animals gave results comparable to those with neutral thermal hydrolysis and/or HCl hydrolysis (Table 2). These data provide validation of this method for the quantitation of Gua in DNA using the IA method described in this paper by demonstrating both the precision and accuracy of this method with several different hydrolysis/digestion methods. We have primarily used enzymatic digestion for three reasons: (1) to avoid the necessity of having to do two

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hydrolyses (since the DNA backbone must undergo acid hydrolysis before the guanine concentration can be determined with neutral thermal hydrolysis), (2) to avoid using high temperatures (since neutral thermal hydrolysis is achieved at 100 °C, formic acid hydrolysis is achieved at 60 °C, and HCl hydrolysis is achieved at 70 °C), and (3) to avoid using acidic conditions. We have recently noted, however, that there appears to be a lowlevel background contaminant released from the IA columns that is converted to Gua during the formic acid hydrolysis that is necessary for conversion of dGuo to Gua when using enzymatic digestion. This background does not appear to be Gua or dGuo, but rather is converted to Gua during processing for GC/MS (data not shown). This level of contamination appears to be very high when columns are first constructed and sharply decreases after several washes of the columns; therefore, the washing step prior to use of the columns is very important. Since the IA columns were made in batch preparations, however, this contaminant could generally be subtracted out for quantitation by running a standard curve along with the samples. This was demonstrated when the same sample processed by enzymatic digestion and neutral thermal hydrolysis gave different analyte to internal standard ratios (0.15 and 0.096, respectively), but gave the same quantitation for Gua (0.086 and 0.089 Gua adducts/108 normal dGuo nucleosides or Gua bases, respectively). This background is not seen when neutral thermal hydrolysis or HCl hydrolysis was used, and these hydrolysis methods may therefore be preferable to enzymatic digestion, particularly for low-level, endogenous samples where this background contamination may most likely interfere. Gua has been detected in tissues of rats exposed to vinyl chloride and in unexposed tissues of mice, rats, and humans (13, 19, 29, 30). In addition, our lab has recently shown that Gua is formed from the reaction of deoxyguanosine with 4-hydroxynonenal and ethyl linoleate under peroxidizing conditions (18). A sensitive and selective method for the detection of this adduct is essential for understanding the role that this adduct may play in carcinogenesis. This method will also allow us to compare the amount of endogenously formed Gua with that formed exogenously in the same sample. This can be accomplished by administering compounds that form Gua that are stably isotopically labeled, which result in Gua with a mass greater than that of endogenous Gua and thereby can be distinguished by GC/MS. The increased sensitivity and selectivity of this method make this type of experiment much more feasible since less DNA is needed for analysis of endogenous Gua.

Acknowledgment. This work was supported by NIEHS Grants ES05779, ES07017, ES07126, and ES05948. We thank Drs. Ten-Yang Yen and Nadia I. Christova-Gueorguieva for the preparation and LC/MS analysis of the CEO-treated rat liver DNA sample and Dr. Ramiah Sangaiah for the synthesis of chemical standards.

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