Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC–ES-MS

Daniel R. Doerge2, Mona I. Churchwell, M. Matilde Marques1 and Frederick A. Beland

National Center for Toxicological Research, Jefferson, AR 72079, USA and
1 Instituto Superior Tecnico, P-1049-001 Lisboa, Portugal


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Electrospray mass spectrometry (ES-MS) is a powerful tool for analysis of carcinogen-adducted DNA. In this study, we developed a quantitative isotope dilution method for analysis of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), the principal nucleoside adduct derived from enzymatic hydrolysis of 4-aminobiphenyl (4-ABP)-modified DNA. The method used column switching valves to perform on-line sample concentration and cleanup, which permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography (LC). ES-MS detection was performed using a single quadrupole instrument by monitoring M+H+ and two fragment ions characteristic for dG-C8-4-ABP, along with M+H+ and a fragment ion for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was ~10 pg on-column, equivalent to 0.7 dG-C8-4-ABP adducts in 107 normal nucleotides for a sample containing 100 µg DNA. The method was applied to the analysis of calf thymus DNA modified in vitro through reaction with N-hydroxy-4-ABP and of hepatic DNA isolated from mice treated in vivo with two dose levels of 4-ABP.

Abbreviations: 4-ABP, 4-aminobiphenyl; ACN, acetonitrile; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; dG, 2'-deoxyguanosine; dG-C8-4-ABP, N-(deoxyguanosine-8-yl)-4-aminobiphenyl; ELISA, enzyme-linked immunosorbent assay; ES, electrospray; ethenoGua, N-2,3-ethenoguanine; Gua, guanine; IAC, immunoaffinity chromatography; M1G, 3-(2-deoxy-ß-D-erythro-pentofuranosyl)-pyrimido[1,2-{alpha}]purin-10(3H)-one; MS, mass spectrometry; NICI, negative ion chemical ionization; RIA, radioimmunoassay; SIR, selected ion recording.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The measurement in tissues from animals and humans of DNA adducts derived from environmental and endogenous carcinogens is essential for relating exposure with DNA damage and, ultimately, with the risk of tumorigenic outcome (1). Because levels of DNA adduction are typically in the range of 1 in 109 to 1 in 106 normal nucleotides, highly sensitive techniques are required for the analysis of small amounts of DNA (1–100 µg) available in many human studies. The method most widely used for the analysis of DNA adducts is the 32P-post-labeling assay (2). This assay uses {gamma}-32P-labeled adenosine triphosphate to incorporate a highly radioactive reporter group into nucleotides derived from enzymatic hydrolysis of DNA. Modified nucleotides are then separated from normal nucleotides by using TLC or HPLC and visualized using radioautography or in-line scintillation counting, respectively. These procedures can yield very sensitive detection of adducted nucleotides, albeit with minimal structural information, and extensive method validation is required for reliable quantitative performance. Immunoassay techniques [Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)] have also been used for DNA adduct analysis (35). Although these assays can be highly sensitive, immunoassay results are limited by the crossreactivity of antisera, and they cannot characterize the adducts chemically. This is a special problem in DNA adduct analysis where the relevant adduct must be determined in the presence of >105-fold excess of unmodified bases. Furthermore, immunoassays usually cannot distinguish completely between nucleoside/nucleotide adducts derived from DNA and RNA (3,5).

In recent years, mass spectrometry (MS), especially electrospray (ES), has proven to be useful for the structure determination of a variety of carcinogen-modified DNA adducts (for a recent review see ref. 6). However, a major remaining challenge is widespread application to the quantitative analysis of DNA adducts derived from exogenous chemicals (7), as well as endogenous metabolic products (8), that are present at trace levels in DNA from humans and animals. We have used ES-MS techniques to validate, through characterization of nucleotide substrates and products, a sensitive 32P-post-labeling HPLC assay for quantification of 3-(2-deoxy-ß-D-erythro-pentofuranosyl)-pyrimido[1,2-{alpha}]purin-10(3H)-one (M1G), the predominant adduct formed in human and animal tissues, produced by reaction of malondialdehyde with DNA (9). This kind of validation permits optimization of several critical assay parameters and can significantly enhance the reliability of DNA adduct measurements. The MS methods most often applied to direct quantitative analysis of DNA adducts are gass chromatography (GC) with negative ion chemical ionization mass spectrometry (GC–NICI-MS) and liquid chromatography with electrospray mass spectrometry (LC–ES-MS) for isotope dilution analysis of modified bases produced by acid hydrolysis of adducted nucleosides. Lin et al. (10) used GC–NICI-MS for analysis of 4-aminobiphenyl (4-ABP) released by alkaline hydrolysis from adducted human bladder and lung DNA at levels of 0.3–50 adducts in 108 normal bases from 1 mg DNA. Rouzer et al. (11) used GC–NICI-MS for analysis of the modified base, M1Gua, in human leukocytes at levels of ~5 adducts in 108 normal bases from 1 mg DNA. This group also used LC–ES-tandem MS to confirm the presence of M1G in leukocyte DNA (12). Yen et al. (13) used LC–ES-MS for the analysis of N-2,3-ethenoguanine (ethenoGua), an adducted base formed by reaction of vinyl chloride with DNA, at levels of ~2 adducts in 108 normal nucleotides from 7.4 mg human liver DNA. However, a disadvantage of these previous MS methods that measured either the hydrolyzed carcinogen or a modified base is that they cannot distinguish between adducts derived from target tissue DNA and those from possible protein and/or RNA contaminants.

Preparation of DNA adducts from small amounts of tissues is a critical component of overall method performance that can often limit ultimate MS detection, especially at the very low levels of adduction relevant to chemical carcinogenesis in humans and animals. In order to achieve high MS sensitivity detection, it is essential to perform efficient adduct purification and enrichment beyond the procedures used to isolate cellular DNA and prepare enzymatic or chemical digests. For example, liquid–liquid partitioning (10), solid-phase extraction (12,13) and immunoaffinity chromatography (IAC) (11) are required for quantification of DNA adducts as the hydrolyzed carcinogen or modified bases. However, these cleanup procedures can be laborious, introduce measurement errors, cause significant analyte losses, and lead to hydrolysis of the modified nucleosides to bases.

Tobacco smoke contains 4-ABP, an arylamine that is carcinogenic in experimental animals and is implicated in the etiology of bladder cancer in cigarette smokers (1,10). The mechanism for arylamine-induced carcinogenesis involves metabolic activation, via N-hydroxylation, to reactive electrophiles that bond covalently with DNA. The principal DNA adduct formed from metabolic activation and covalent bonding of 4-ABP to DNA is N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). In this study, crude enzymatic DNA hydrolysates were processed using LC with column switching to perform sample purification and enrichment of the modified nucleoside prior to separation using a narrow-bore LC column. Detection with high sensitivity and quantification were achieved using isotope dilution ES-MS with selected ion recording (SIR) of three ions characteristic for the specific dG-C8-4-ABP adduct and two ions for a deuterated internal standard. These procedures were used for analysis of calf thymus DNA modified in vitro by several levels of N-hydroxy-4-ABP, and for analysis of hepatic DNA isolated from two groups of mice who had received single doses of 4-ABP.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sample preparation
DNA modified in vitro. Calf thymus DNA in 10 mM sodium citrate buffer, pH 5.0, was purged with argon and reacted overnight in ethanol with several stoichiometric ratios of N-hydroxy-4-ABP:DNA nucleosides (1.85x10–5, 1.85x10–4 and 1.85x10–3). The solution was extracted sequentially with phenol and n-butanol and, following precipitation with ethanol and sodium chloride, the DNA was isolated by centrifugation, and dissolved in 5 mM bis-Tris, 0.1 mM EDTA buffer, pH 7.1. (For complete details see ref. 14.)

DNA modified in vivo. Eight-week-old male B6C3F1 mice (three groups of four animals) were each injected i.p. with either 0.1 mg 4-ABP, 1.0 mg 4-ABP or solvent. After 24 h, the mice were killed, livers were pooled by group, and DNA was prepared from isolated nuclei. The samples were dissolved in 5 mM bis-Tris, 0.1 mM EDTA buffer, pH 7.1. (For complete details see ref. 14.)

Preparation and characterization of dG-C8-4-ABP-d9 internal standard. The labeled internal standard was prepared from biphenyl-d10 (Aldrich, Wilwaukee, WI) by nitration with ammonium nitrate in the presence of trifluoroacetic anhydride, followed by reduction to N-hydroxy-4-ABP-d9, and O-acetylation with acetyl cyanide. This product was reacted with deoxyguanosine (dG) and the adduct was purified on Sephadex LH-20 (Pharmacia/PL Biochemicals, Piscataway, NJ) using a 20–80% step gradient of aqueous methanol (14). The adduct eluted at 70% methanol and was quantified using UV spectrophotometry (15). The concentration of dG-C8-4-ABP standards (unlabeled and d9) was determined spectrophotometrically using an extinction coefficient of 31 mM–1 at 305 nm (14). Analysis of the dG-C8-4-ABP-d9 standard using ES-MS showed that it was ~99% pure and contained <0.1% of dG-C8-4-ABP-H9 (data not shown). A calibration plot of response ratios (LC–ES-MS integrated peak area ratio for M+H+ ions H9/d9) versus concentration ratios (0–10) was constructed for injection of standards (varying amounts of H9, 0–1.25 ng, and a constant amount of d9, 125 pg). The plot was linear with a correlation coefficient of 0.999 and a slope of 0.93.

Enzymatic hydrolysis of DNA. After isolation of in vitro- and in vivo-modified DNA, aqueous buffered solutions of modified DNA in 100 µl total volume were spiked with the dG-C8-4-ABP-d9 internal standard at approximately the level of dG-C8-4-ABP expected in the sample (117–293 pg d9). This was possible because estimates of adduct levels were available from other techniques (14). The DNA was hydrolyzed enzymatically to nucleosides by treatment with DNase I, followed by alkaline phosphatase and snake venom phosphodiesterase (14), and the entire sample was injected into the LC–ES-MS system described below.

Liquid chromatography
On-line sample preparation was performed using a liquid handling system composed of a manual injector (model 7125; Rheodyne, Cotati, CA) or an autosampler (AS3500; Dionex, Sunnyvale, CA), two automated switching valves (TPMV; Rheodyne), and two HPLC pumps [a Dionex GP40 quaternary gradient pump and an ISCO 260D syringe pump (ISCO, Lincoln, NE)]. Switching valve 1 allowed the gradient pump eluent to either load a sample onto the trap column and then wash it (dashed line configuration shown in Figure 1Go) or back flush the trap column contents onto the analytical column (not shown). Switching valve 2 was used to divert the trap column effluent to either waste or to the analytical column. The gradient pump was used for sample injection, cleanup, elution, analysis and regeneration of the trap and analytical columns, while the syringe pump containing 65% H2O/35% acetonitrile (ACN) was used to equilibrate the analytical column and keep a constant flow of mobile phase going into the mass spectrometer during sample loading and preparation periods (solid line configuration shown in Figure 1Go).



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Fig. 1. Schematic diagram of switching valves used for on-line sample preparation. The original configuration is shown for the sample loading/washing phase in which the gradient pump eluent goes through valves 1 and 2 into the trap column and then to waste (dashed lines). During this interval, the syringe pump provided mobile phase to the analytical column and the mass spectrometer (solid lines). After sample loading and washing were complete, both valves were switched and the gradient pump eluent back flushed the trap column to elute the analytes onto the analytical column and into the mass spectrometer. During this time, the syringe pump eluent was diverted to waste.

 
The sample was loaded for 3 min at 1 ml/min with 100% H2O onto a reverse-phase trap column (POROS 10-R2, 2.1x30 mm; PerSeptive Biosystems, Framingham, MA) and then the trap column was washed with 70% H2O and 30% MeOH for 3 min at 1 ml/min to waste. After switching both valves, the concentrated sample zone was back flushed from the trap column onto the analytical column (Columbus C18, 2x150 mm, 5µ; Phenomenex, Torrance, CA) at 0.2 ml/min with 65% H2O/35% ACN and sample components were eluted into the mass spectrometer. When the 11 min run was finished, a rapid gradient ramp up to 90% ACN in 5 min was used to clean both the trap and analytical columns. Finally, the mobile phase was switched back to 35% ACN in a 5 min gradient step. Then both valves were switched to their initial positions to equilibrate both the trap and analytical columns at the starting mobile-phase composition and the process was repeated. It should be noted that all valve switching was done under computer control from the LC gradient pump software. In order to prevent analyte carryover, both sides of the manual injector were washed after each analysis.

Mass spectrometry
A Platform II single quadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with an ES interface was used with an ion source temperature of 150°C. Positive ions were acquired in SIR mode (dwell time, 0.3 s; span, 0.02 Da; interchannel delay time, 0.03 s) for the M+H+ and BH2+ ions of both dG-C8-4-ABP-H9 and dG-C8-4-ABP-d9, along with a third confirmatory fragment ion derived from dG-C8-4-ABP-H9. The sampling cone-skimmer voltage was switched between 20, 70 and 100 V to produce in-source collision-induced dissociation in concert with acquisition of the respective selected ion (16). The ions acquired and the optimized cone voltages are shown in Table IGo.


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Table I. Selected ion acquisition parameters for dG-4-ABP-H9, d9
 

    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Our goal was to develop a quantitative method for analyzing the major nucleoside adduct from 4-ABP-modified DNA, dG-C8-4-ABP. Experimental constraints made it essential that the method be highly sensitive (detection limit <50 fmol on-column) and specific for dG-C8-4-ABP, use small amounts of DNA <=100 µg), require minimal sampling handling, have the potential for automation, and perform with adequate recovery, precision and accuracy. In initial experiments, the use of butanol extraction or solid-phase extraction with C18 cartridges was evaluated as a means to purify and concentrate the dG-C8-4-ABP present in DNA samples. In both cases, very low recovery (0–20% for calf thymus DNA 3; Table IIGo) was observed for either dG-C8-4-ABP from in vitro-modified DNA hydrolysates or the added internal standard.


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Table II. Isotope dilution quantification of dG-C8-ABP in DNA modified in vitro and in vivo
 
To achieve the stated goals, enzymatically hydrolyzed DNA, containing dG-C8-4-ABP and excess unmodified bases, was first spiked with an equivalent amount of d9 internal standard. The DNA digest was introduced into a liquid handling system comprised of two pumps, an injection valve, two six-port automated switching valves, a reverse-phase trap column, and a high resolution reverse-phase narrow-bore analytical LC column. It should be noted that the buffered aqueous solution containing enzymes and hydrolyzed DNA was injected directly. The trap column was used for sample cleanup and concentration because it selectively retained the relatively non-polar dG-C8-4-ABP but not the large excess of polar unmodified nucleosides. The POROS perfusion chromatographic medium was chosen because of the high flow rates and rapid equilibration rates possible that lead to shorter sample loading and washing times (17). The flow through the trap column was then reversed and the dG-C8-4-ABP and deuterated internal standard were eluted onto the analytical column in a narrow band. Compounds were subsequently eluted into an ES interface with a single quadrupole mass spectrometer and a multiple ion acquisition method was used to quantify H9 and d9 isotopomers. Under these conditions, recovery of the deuterated internal standard averaged 72 ± 16% (mean ± SD, range 43–107, n = 25). The losses in analyte appeared to be effectively compensated for by corresponding losses in the internal standard because no association between dG-C8-4-ABP-H9 determination and recovery of the dG-C8-4-ABP-d9 was noted (not shown).

Another advantage to the sample preparation methodology described here is that, if needed, additional specificity can be gained through use of on-line immunoaffinity cleanup for DNA adducts. In a previous paper, we described use of automated on-line IAC and LC–ES-MS detection and quantification of fumonisins and related compounds in corn products (18). The heart of this procedure is an immunoaffinity cartridge made by covalently coupling antibody to perfusion chromatography media. This coupling procedure gave a durable IAC matrix that retained full function despite repeated cycling of the acidic and organic solvents used to wash and elute analytes from the cartridge.

Full scan mass spectra for dG-C8-4-ABP were acquired from an authentic dG-C8-4-ABP-H9 standard at several different sampling cone-skimmer potentials. At low cone voltage (20 V), formation of the protonated molecule of dG-C8-4-ABP predominated. As the voltage was increased, formation of the respective BH2+ ion occurred. As the voltage was increased further, additional fragmentation of the BH2+ ion occurred (see Figure 2Go for the 100 V spectrum and some proposed fragmentation reactions). This spectral information was used to optimize the SIR acquisition method by including M+H+, BH2+ and another confirmatory fragment ion (m/z 195) derived from dG-C8-4-ABP (Table IGo). The same cone voltages were used for monitoring the M+H+ and BH2+ ions from the d9 internal standard. Figure 3Go shows the selected ion chromatograms for an injection of 250 pg each of dG-C8-4-ABP-H9 and the deuterated internal standard.



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Fig. 2. In-source collision-induced dissociation of dG-C8-4-ABP. The full scan mass spectrum (m/z 100–500) was acquired at a sampling cone-skimmer voltage of 100 V from 300 ng of dG-C8-4-ABP injected on-column using LC–ES-MS. The inset shows the proposed fragments acquired in the MS–SIR method.

 


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Fig. 3. Selected ion chromatograms for dG-C8-4-ABP-H9 and dG-C8-4-ABP-d9 standards. Selected ions for the M+H+ (m/z 435 and m/z 444), BH2+ (m/z 319 and m/z 328) and m/z 195 ions were acquired as shown in Table IGo from an injection containing 250 pg each of the H9 and d9 isotopomers. The retention time (in min) is listed above the major peaks and the respective selected ion traces are labeled at the right.

 
Isotope dilution calibration curves for concentration ratios versus measured response ratios of dG-C8-4-ABP-H9 and dG-C8-4-ABP-d9 were generated for both the M+H+ and BH2+ ions. Both plots were highly linear and the slopes were used to calculate the amount of dG-C8-4-ABP present in the DNA hydrolysates. In addition, this method also made it possible to quantify dG-C8-4-ABP even if a matrix component interfered with one of the two ions monitored for unlabeled or deuterated isotopomers. Typically, no such interference was observed and the computed values obtained from M+H+ responses agreed closely with those from BH2+ in all samples. Therefore, only the values for M+H+ are shown. The amount of dG-C8-4-ABP-d9 added to each sample varied with the anticipated amount of dG-C8-4-ABP-H9 to make the response ratio as close to one as possible; however, no less than 117 pg (270 fmol) of dG-C8-4-ABP-d9 was injected. By monitoring the molecular ion and two additional fragment ions in the MS–SIR acquisition method, a significant measure of structure proof not available from a single ion acquisition was provided; moreover, this added structural information provided by acquisition of two fragment ions was achieved without sacrificing overall analytical sensitivity (data not shown).

Results from quantitative analysis of dG-C8-4-ABP present in the DNA samples are presented in Table IIGo. Using an authentic dG-C8-4-ABP-H9 standard, the detection limit (signal to noise ratio of ~3) was estimated to be 23 fmol (10 pg) through the entire on-line sample preparation analogous to that performed on a hydrolysate of 100 µg DNA (data not shown). This is equivalent to 0.23 fmol/µg DNA or 0.73 dG-C8-4-ABP adducts in 107 normal nucleotides. Analysis of control DNA from either calf thymus (Figure 4Go) or untreated-mouse liver (Figure 5Go) consistently showed no responses for dG-C8-4-ABP-H9 even though typical recoveries of the deuterated internal standard were observed. Samples containing 50–100 µg of calf thymus DNA that had been modified in vitro to three different substitution levels through reaction with N-hydroxy-4-ABP were analyzed and quantified (Figure 4Go; Table IIGo). The substitution levels ranged from 1.8 to 430 adducts in 107 normal nucleotides. The intra-assay precision for samples modified at the three levels varied from 6.1–25.1% relative standard deviation. Hepatic DNA samples (50–100 µg), obtained from mice that had been administered a single dose of 0.1 or 1.0 mg 4-ABP, were also analyzed and quantified using this technique (Figure 5Go; Table IIGo). The substitution level for these in vivo-modified samples corresponded to ~4.9 and 30 dG-C8–4-ABP adducts in 107 normal nucleotides from the respective groups of mice. The intra-assay precision varied from 9.7 to 17.7% relative standard deviation. The inter-assay precision was determined by analyzing the 1.0 mg 4-ABP-treated mouse hepatic DNA on two different days (Table IIGo). The respective average values agreed within experimental error (8.6 and 9.8 fmol/µg DNA) and the precision was similar (9.7 and 11.8% relative standard deviation).



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Fig. 4. Selected ion chromatograms for the M+H+ and BH2+ ions from dG-C8-4-ABP-H9 and dG-C8-4-ABP-d9 in control calf thymus DNA and in vitro-modified calf thymus DNA samples. The internal standard was added to DNA solutions from control (d9 = 117 pg, left panels) and in vitro-modified calf thymus DNA (stoichiometric ratio 1.85x10–3 N-hydroxy-4-ABP/DNA nucleotides, d9 = 253 pg, right panels), followed by enzymatic hydrolysis and analysis using LC–ES-MS as described in the legend to Figure 3Go.

 


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Fig. 5. Selected ion chromatograms for the M+H+ and BH2+ ions from dG-C8-4-ABP-H9 and dG-C8-4-ABP-d9 in control mouse hepatic DNA and in vivo-modified mouse hepatic DNA samples. The internal standard dG-C8-4-ABP-d9 (117 pg) was added to DNA solutions from untreated mouse hepatic DNA (left panels) and in vivo-modified mouse hepatic DNA (0.1 mg treatment group, right panels) followed by enzymatic hydrolysis and analysis using LC–ES-MS. The integrated peak areas used for isotope dilution quantification are listed below the retention time (in min) above the respective peaks.

 
We recently reported on a comparative interlaboratory quantitative analysis of the same calf thymus and mouse liver DNA samples described here using different adduct detection methodologies (14). In that study, analyses of in vitro-modified calf thymus DNA and in vivo-modified mouse hepatic DNA using 32P-post-labeling, DELFIA and the described LC–ES-MS assay were compared with the total binding of radiolabeled N-hydroxy-4-ABP. Although all methods had adequate sensitivity for these samples, the results from 32P-post-labeling consistently underestimated adduct content (1–5%), DELFIA consistently overestimated adduct content (101–534%) and the LC–ES-MS results agreed most closely with the specific radiolabel binding. The statistical performance (relative standard deviation) of the LC–ES-MS method was also superior to the other methods for analysis of in vitro- and in vivo-modified DNA. For example, LC–ES-MS analysis of adduct content in 0.1 mg 4-ABP-treated mouse hepatic DNA had a relative standard deviation of 18% and for DELFIA analysis, it was 31–36%. These findings suggest that when adequate sensitivity can be achieved, there is significant added value in terms of accuracy and precision from using LC–ES-MS for quantitative analysis of DNA adducts.

We also showed that the results from LC–ES-MS quantification of carcinogen binding to standard DNA samples can be used as a benchmark to effectively calibrate analyses from other quantitative methods (e.g. 32P-post-labeling or immunochemical) for interlaboratory validation procedures (14). Moreover, the degree of chemical characterization of adducts through ES-MS acquisition of several ions, including molecular and structurally significant fragments, is not possible using these other techniques. Finally, the use of LC/MS with column switching, and its potential for automation, provides significant improvement in the ease of DNA adduct analysis relative to either 32P-post-labeling, which requires handling large amounts of a highly active radioisotope, or immunochemical methods, which require extensive intra-assay calibration procedures.

The remaining challenge will be to apply this methodology to the study of dG-C8-4-ABP adducts in human tissues.


    Acknowledgments
 
We thank Drs Keith Newkirk and Dean Roberts, NCTR, for assistance with column packing and helpful discussions.


    Notes
 
2 To whom correspondence should be addressed Email: ddoerge{at}nctr.fda.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

  1. Poirier,M.C. and Beland,F.A. (1997) Aromatic amine DNA adduct formation in chronically exposed mice: considerations for human comparison. Mutat. Res., 376, 177–184.[ISI][Medline]
  2. Beach,A.C. and Gupta,R.C. (1992) Human biomonitoring and the 32P-post-labeling assay. Carcinogenesis, 13, 1053–1074.[ISI][Medline]
  3. Roberts,D.W., Benson,R.W., Groopman,J.D., Flammang,T.J., Nagle,W.A., Moss,A.J. and Kadlubar,F.F. (1988) Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl. Cancer Res., 48, 6336–6342.[Abstract]
  4. Schoket,B., Doty,W.A., Vincze,I., Strickland,P.T., Ferri,G.M., Assennato,G. and Poirier,M.C. (1993) Increased sensitivity for determination of polycyclic aromatic hydrocarbon–DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Cancer Epidemiol. Biomarkers Prev., 2, 349–353.[Abstract]
  5. Poirier,M.C. (1997) DNA adducts as exposure biomarkers of cancer risk. Environ. Health Persp., 105 (Suppl. 4), 907–912.
  6. Esmans,E.L., Broes,D., Hoes,I., Lemiere,F. and Vanhoutte,K. (1998) Liquid chromatography–mass spectrometry in nucleoside, nucleotide, and modified nucleotide characterization. J. Chrom. A, 794, 109–127.[ISI]
  7. Beland,F.A. and Kadlubar,F.F. (1990) Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons. In Cooper,C.S. and Grover,P.L. (eds) Handbook of Experimental Pharmacology. Chemical Carcinogenesis and Mutagenesis. Springer-Verlag, Heidelburg, Germany, pp. 267–325.
  8. Marnett,L.J. and Burcham,P.C. (1993) Endogenous DNA adducts: Potential and paradox. Chem. Res. Toxicol., 6, 771–785.[ISI][Medline]
  9. Yi,P., Sun,X., Doerge,D.R. and Fu,P.P. (1998) An improved 32P-post-labeling/HPLC method for the analysis of malonaldehyde-derived 1,N2-propanodeoxyguanosine DNA adduct in animal and human tissues. Chem. Res. Toxicol., 11, 1032–1041.[ISI][Medline]
  10. Lin,D., Lay,J.O.Jr, Bryant,M.S., Malaveille,C., Friesen,M., Bartsch,H., Lang,N.P. and Kadlubar,F.F. (1994) Analysis of 4-aminobiphenyl–DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography/mass spectrometry. Env. Health Perspect., 102 (Suppl. 6), 11–16.
  11. Rouzer,C.A., Chaudhary,A.K., Nokubo,M., Ferguson,D.M., Reddy,G.R., Blair,I.A. and Marnett,L.M. (1997) Analysis of the malondialdehyde-2'-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/negative chemical ionization/mass spectrometry. Chem. Res. Toxicol., 10, 181–188.[ISI][Medline]
  12. Chaudhary,A.K., Nokubo,M., Reddy,G.R., Yeola,S.N., Morrow,J.D., Blair,I.A. and Marnett,L.J. (1995) Characterization of endogenous DNA adducts by liquid chromatography/electrospray ionization tandem MS. J. Mass Spectrom., 30, 1157–1166.[ISI]
  13. Yen,T.Y., Christova-Gueoguieva,N.I., Scheller,N., Holt,S., Swenberg,J.A. and Charles,M.J. (1996) Quantitative analysis of the DNA adduct N2,3-ethenoguanine using LC/ESI-MS. J. Mass Spectrom., 31, 1271–1276.[ISI][Medline]
  14. Beland,F.A., Doerge,D.R., Churchwell,M.I., Poirier,M.C., Schoket,B. and Marques,M. (1999) Synthesis, characterization, and quantitation of a 4-aminobiphenyl DNA adduct standard. Chem. Res. Toxicol., 12, 68–77.[ISI][Medline]
  15. Shapiro,R., Underwood,G.R., Zawadzka,H., Broyde,S. and Hingerty,B.E. (1986) Conformation of d(CpG) modified by the carcinogen 4-aminobiphenyl: A combined experimental and theoretical analysis. Biochemistry, 25, 2198–2205.[ISI][Medline]
  16. Doerge,D.R., Churchwell,M.I., Holder,C.L., Rowe, L. and Bajic,S. (1996) Detection and confirmation of ß-agonists in bovine retina using LC-APCI/MS. Anal. Chem., 68, 1918–1923.[ISI][Medline]
  17. Regnier,F.E. (1991) Perfusion chromatography. Nature, 350, 634–635.[ISI][Medline]
  18. Newkirk,D.K., Benson,R.W., Howard,P.C., Churchwell,M.I., Doerge,D.R. and Roberts,D.W. (1998) On-line immunoaffinity capture coupled with high performance liquid chromatography and electrospray ionization mass spectrometry for automated determination of fumonisins. J. Ag. Food Chem., 46, 1677–1688.[ISI]
Received August 13, 1998; revised January 20, 1999; accepted March 1, 1999.