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Carcinogenesis Advance Access originally published online on August 21, 2006
Carcinogenesis 2007 28(2):342-349; doi:10.1093/carcin/bgl142
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients

Beatriz Zayas1,2, Sara W. Stillwell2, John S. Wishnok2, Laura J. Trudel2,*, Paul Skipper2, Mimi C. Yu4, Steven R. Tannenbaum2,3 and Gerald N. Wogan2

1 Universidad Metropolitana, San Juan, Puerto Rico MA, USA
2 Biological Engineering Division, Massachusetts Institute of Technology, Cambridge MA, USA
3 Chemistry Department, Massachusetts Institute of Technology, Cambridge MA, USA
4 The Cancer Center, University of Minnesota Minneapolis, MN, USA

*To whom correspondence should be addressed. Email: ljtrudel{at}mit.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 109 bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW = 443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444 -> 328. The method was validated by analysis of DNA (100 µg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 µg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5–80 dG-C8-ABP adducts per 109 bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12–1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

Abbreviations: 4-ABP, 4-aminobiphenyl; acetyl cyanide, pyruvonitrile; d9-dG-C8-ABP, deuterated dG-C8-4-ABP; dG-C8-ABP, dG-C8-4-aminobiphenyl; DMAC, dimethylacetamide; GC/MS, gas chromatography-mass spectrometry; LC/MS, liquid chromatography mass spectrometry; LC/MS/MS, liquid chromatography tandem mass spectrometry; SRM, selected reaction monitoring


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Bladder cancer is a disease of continuing public health significance, occurring with high prevalence in men 60 years of age and older in the US. Some known risk factors for bladder cancer development include smoking, occupational exposure, genetic susceptibility, infectious diseases and radiation therapy (1). Smoking and occupational exposure have strongly implicated aromatic amines as being carcinogenic for the bladder (2,3), with 4-aminobiphenyl (4-ABP) being one of the most potent. Exposure to aromatic amines continues to be of concern because it appears that in addition to tobacco smoke, there exist other identified as well as unidentified sources. Identified sources include some commercial hair dyes (4). The nearly ubiquitous presence of hemoglobin (Hb) adducts of aromatic amines in persons without any obvious exposure to the compounds is evidence for the existence of other sources.

Formation of aromatic amine adducts in the DNA of the bladder epithelium is commonly regarded as an important mechanism of action by these compounds (5). Recent evidence for the importance of these adducts comes from a comparison of the mutational hot spots in the p53 gene in human bladder cancer and the adduct spectrum in p53 in human bladder cells exposed to metabolically activated 4-ABP (6,7). Remarkably, codons 175, 248, 273, 280 and 285 are targets both in vivo and in vitro, a powerful argument for the importance of aromatic amines in bladder cancer. Specific evidence for the presence of 4-ABP DNA adducts in human bladder tissue has been presented in a report of the analysis of biopsy samples using a gas chromatography-mass spectrometry (GC/MS) method (8). This study also found that the presence of adducts correlated with tumor grade.

One objective of this study was to develop and optimize a sensitive liquid chromatography mass spectrometric (LC/MS) method for the quantitative analysis of 4-ABP DNA adducts in human bladder tissue. As previously noted (9), LC/MS has been underutilized in assessing human exposure, but this methodology offers the greatest available degree of analyte characterization. Levels of DNA adducts expected from exposure to 4-ABP in tissues of humans range from 1 per 109 to 1 per 106 nucleotides (10), implying the need for highly sensitive methods for detection. Although post-labeling and immunochemical detection methods provide the required sensitivity and quantitative information, they lack the capability to show structural or chemical characteristics of the analyte.

A further objective of this study was to generate additional data regarding the presence of 4-ABP adducts in bladder DNA that might confirm and extend previous findings. To this end, we undertook application of the developed liquid chromatography tandem mass spectrometry (LC/MS/MS) approach to the analysis of a set of human bladder tissues drawn from a distinct population of bladder cancer patients. We did not necessarily expect to establish any causal relationship between the detected DNA adducts in these tissues and the formed tumors because of the temporal disjunction between exposure that might have initiated the tumors and exposure giving rise to adducts present at the time of cystectomy. However, levels of 4-ABP DNA adducts in the tissues when resected might reflect contemporaneous exposure to 4-ABP as suggested by studies (8,11) showing an association of 4-ABP bladder DNA adduct levels with a proxy—smoking status—for 4-ABP exposure. As we have recently reported (12), smoking status and cigarette smoking are imperfect measures that may seriously misrepresent actual exposure to 4-ABP, especially among bladder cancer case subjects. A final objective of this study, then, was to test for the existence of a relationship between 4-ABP bladder DNA adduct level and 4-ABP exposure, using Hb adducts of 4-ABP as a more quantitative measure of exposure than tobacco consumption and one that reflects all sources of exposure.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Caution
The following chemicals are hazardous and should be handled carefully: 4-nitrobiphenyl, ammonium sulfide, dimethylacetamide (DMAC), triethylamine and pyruvonitrile (acetyl cyanide). In addition 4-ABP is a known bladder carcinogen and inhalation or skin contact should be prevented.

Chemicals
DNA extraction chemicals were purchased as follows: RNase (DNase free) and alkaline phosphatase from calf intestine from Roche (Indianapolis, IN, USA); Pronase protease and nuclease P1 from Calbiochem (San Diego, CA, USA); Tris–HCl, EDTA–Na2, equilibrated phenol, phenol/chloroform/isoamyl (25:24:1), 8-hydroxyquinoline, phosphodiesterase 1, Type II from Crotalus Adamanteus from Sigma (St Louis, MO, USA). Nucleosides (2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine and 2'-deoxythymine), Sigmacote (SL-2), calf thymus DNA, salmon testes DNA and human placenta DNA were from Sigma. Chemicals used in the synthesis of dG-C8-4-aminobiphenyl (dG-C8-ABP) were as follows: 4-nitrobiphenyl, ammonium sulfide (20% water); DMAC; triethylamine; and pyruvonitrile, all from Sigma-Aldrich.

dG-C8-ABP analyte and deuterated internal standard
The dG-C8-ABP standard was generated in-house by modifications of previously reported procedures (13,14). Briefly, N-OH-4-ABP was generated by reaction of 4-nitrobiphenyl (dissolved in dimethylacetamide) with ammonium sulfide for ~3 h at 0°C. The yellow precipitate was extracted with ether and the organic layer washed with water and saturated NaCl. The organic layer was dried with magnesium sulfate and filtered. The residue was dissolved in methylene chloride and kept at –20°C overnight. The bright yellow precipitate (N-OH-4-ABP) was dried in vacuo.

N-OH-4-ABP dissolved in anhydrous ether was reacted (at 0°C) with triethylamine and pyruvonitrile. The mixture was stirred for 30 min, the cold bath removed and the yellow mixture, including precipitate, was extracted with methylene chloride followed by an additional wash with ice water. The organic layer containing N-O-acetyl-4-ABP was reacted with a warm (55–60°C) 2'-deoxyguanosine (dG) solution in ethanol/water (2/1). The resulting mixture was stirred for 1 h at 55–60°C, extracted with water, concentrated to dryness and purified by flash chromatography (25 g silica gel, packed with methylene chloride). Prior to loading, the sample was dissolved in 10% methanol/methylene chloride. The resulting product was dG-C8-ABP.

The crude product was further purified by reverse phase HPLC analysis, monitored at 300 nm with a Beckman 166 spectrophotometric detector (Beckman Coulter, Inc., Fullerton, CA, USA) coupled to a 125 solvent module consisting of two pumps. Flow rate was at 1.5 ml/min, with a Phenomenex C18, 4.6 mm x 250 mm column. Solvents were A: high purity water and B: methanol. Concentration was determined by UV absorbance at 300 nm using an extinction coefficient of 31 000. Final characterization of the dG-C8-ABP was determined by electrospray mass spectrometry, with monitoring of the [M+H+]+1 ion at m/z 435.1. Additional fragmentation of the [M+H+]+1 ion to m/z 319.1 was also monitored. This non-deuterated compound was used for optimization of the immunoaffinity recovery method and calibration curve.

A deuterated internal standard, deuterated dG-C8-4-ABP (d9-dG-C8-ABP), was obtained from Dr Frederick Beland of the National Center for Toxicological Research, Jefferson, AR, USA. In each DNA enzymatic hydrolysis, a known amount of the internal standard (56–282 fmol) was added to the reaction mixture prior to the first incubation stage.

Study population
Urinary bladder tissues were obtained from 37 patients with bladder cancer, during their bladder surgery at the University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA, USA. Information including age, gender, race and smoking status was obtained through in-person interviews. Tumor stage was abstracted from patients' pathology reports. The Institutional Review Board at the University of Southern California had approved this study.

DNA from tumor and surrounding ‘non-tumor’ tissue of 37 subjects was isolated by phenol/chloroform extraction. Yield of DNA from tumors (60–2000 µg) was higher than that from non-tumor tissues (20–400 µg). Since prior studies have shown that DNA adduct levels in human tissues tend to be in the order of 1 adducted base in 109 nucleosides or less, and given the limited information available for adduct levels in human bladder tissue, we established 200 µg of DNA as the minimum amount for analysis in order to assure adduct detection when present. By this criterion, samples from 27 out of 37 subjects yielded sufficient DNA from tumor tissue to permit analysis. Sufficient DNA was recovered from non-tumor tissues of 14 of the 27 subjects (52%) to permit analysis of both tumor and non-tumor tissues in the same subject. Of the 27 subjects, 5 (18.5%) were female and 22 (81.5%) were male; the age distribution was from 47 to 69 years. Racial distribution was 21 white (78%), 3 black (11%), 2 Hispanic (7%) and 1 Asian (4%). Tumor stage profiles included 10 stage-4 (37%), 15 stage-3 (55%) and 2 stage-0 (7%). The group included one smoker (4%), 25 non-smokers (93%) and one of unknown status. Since only one subject was identified as a current smoker, results are presumed to reflect adduct levels in ex- or non-smokers.

At the time of sample preparation and analysis, none of the authors at MIT had knowledge of the subject population demographics or lifestyle. Information regarding gender, age, ethnicity and tumor stage (Table I) was disclosed after analyses had been completed.


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  		Table I Demographics of the study population, dG-C8-ABP adduct levels in bladder DNA and 4-ABP Hb adduct levels in blood

 
DNA isolation—human urinary bladder tissues
Tissue samples were cut into 100 mg portions that were placed in a conical tube and combined with 1.5 ml of 1x TE homogenization buffer (500 mM Tris–HCl, pH 8.0 and 100 mM EDTA–Na2). Twenty microliters of 10% SDS and 1 ml protease (10 µg/µl) were added and incubated at 37°C for 24 h, after which tissues were homogenized using a Polytron at 1.5 speed on ice for 5–10 s. Following disruption, 22 µl of RNase (0.5 µg/µl, DNase free) were added and incubated for 2 h at 37°C. DNA was then isolated by a series of phenol–chloroform extractions. In the first extraction, homogenized tissue (1.5 ml) was combined with an equal volume of TE-saturated phenol containing 0.1% 8-hydroxyquinoline, mixed by inversion for 5 min, and centrifuged at 9000 r.p.m. for 10 min. The extraction procedure was repeated twice as above, but with centrifugation for 5 min and 3 min, respectively. DNA was precipitated from the final aqueous phase by combining 150 µl of 5 M NaCl and 1.5 ml cold ethanol, maintained at –20°C for 2 h and centrifugation at 9000 r.p.m. for 7 min. The resulting DNA pellet was rinsed with 1.5 ml 70% ethanol, recentrifuged, dissolved in 1 ml water and sheared by repeated passage through a small bore needle prior to storage at –20°C. DNA concentration was calculated from absorbance at 260 nm (1 OD = 50 µg/ml), and purity by UV ratios of 260/280 nm (1.7–2.0 OD indicating low RNA and protein contamination).

Rat liver DNA
Liver DNA extracted from 4-ABP-treated and untreated rats was used as positive and negative controls. Four adult female Fischer rats (Charles River Laboratory, Wilmington, MA, USA) were maintained in an AAALAC accredited facility, individually housed and fed ad libitum. Rats were held for 7 days after arrival to allow acclimation. Animals were injected intraperitoneally with 1.25 mg/100 µl DMSO of either 4-ABP or 1 mCi 3H4-ABP; the control received 100 µl of DMSO. Twenty-four hour post-treatment, animals were killed by CO2 asphyxiation and livers removed, washed with PBS, dried, quick frozen in liquid nitrogen and stored at –100°C. DNA was then isolated using the extraction protocol used for the human tissues as described above.

Pig urinary bladder DNA
Since the structure of the porcine urinary bladder closely resembles that of the human, DNA from this source was used in the optimization of the analytical method. Bladder tissue was obtained through the MIT Tissue Harvest program. Post mortem, the bladder from an untreated potbelly pig was surgically removed, washed in PBS, dried, cut into 200 mg aliquots and frozen. DNA was extracted as above.

Enzymatic DNA digestion
DNA was enzymatically hydrolyzed to nucleosides. To ensure complete digestion of the valuable human DNA, incubation time and temperature, enzyme concentrations and reaction volume were optimized using DNA samples from pig bladder, calf thymus or salmon testis. Co-chromatography of digestion products with 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine and 2'-deoxythymidine standards was used to identify analytes. Digestion for 2 h at 37 or 40°C, as used in some previously reported methods, was found to result in the presence of unknown peaks, whereas incubation for a total of 6 h (2 periods of 3 h) at 37°C resulted in complete digestion. Reverse phase HPLC analysis was carried out with monitoring at 260 nm, at a flow rate of 1 ml/min using a Phenomenex Luna C18, 5 µ analytical column (250 mm x 4.60 mm). Solvents were A: 0.1 M ammonium acetate and B: acetonitrile. Samples were eluted with a 40 min non-linear gradient from 0 to 15% B in 20 min followed by a 10 min linear gradient to 30% B for duration of 5 min.

The optimized DNA digestion procedure is as follows: 200 µg DNA in water (100 µl) was placed in a polystyrene tube with snap cap, 8 U nuclease P1 (20 µl in water) was added, and the pH adjusted with 20 µl 1 M NaOAC, pH 5.1, and 20 µl 2 mM ZnCl2. A known amount of deuterated dG-C8-ABP internal standard was added at this step. After incubation for 3 h at 37°C, 20 µl of 1 M carbonate buffer (1 M Na2CO3 and 1 M Na2HCO3) pH 9; 0.05 U phosphodiesterase I Type II and 4 U alkaline phosphatase were added, mixed and incubated again for 3 h at 37°C. The final volume of the reaction was ~200 µl. After incubation, samples were treated with monoclonal antibodies as described below.

Immunoaffinity recovery of analyte and internal standard
Monoclonal antibody (3D6) specific for dG-C8-ABP was bound to cyanogen bromide-activated Sepharose at 4 mg/ml gel as reported previously (15). Prior to tissue sample processing, length of the sample/antibody incubation time, amount of antibody gel and methanol elution volumes were optimized by determining the recovery of dG-C8-ABP standard by UV absorbance at 305 nm in methanol. Optimal recovery was achieved with columns containing 50–500 µl of antibody gel. Sepharose-bound antibody was stored in PBS containing sodium azide at 4°C, and prior to use, the gel was rinsed with PBS then water. Incubation periods of sample and antibody gel in 1 ml Silanized glass vials ranged from 3 to 24 h at 4°C. Samples were then loaded into an empty 5 ml chromatography cartridge (Bio-Rad, CA, USA), for washing and elution by gravity. The optimized method produced over 90% recovery of dG-C8-ABP after overnight incubation at 4°C of 200 µl enzymatic digest with an equal volume of antibody gel, followed by elution with 500 µl methanol.

Analysis was carried out on 100 µg of digested DNA samples, spiked as appropriate with a known amount of d9 internal standard. Samples were loaded into a chromatographic cartridge, enzymes and salts were removed by washing with 20 ml water, then adduct was eluted with 500 µl methanol. The methanol eluant was brought to dryness in vacuum for 30 min, after which samples were stored for no longer than 3–4 days; they were redissolved in 20 µl methanol for LC/MS/MS on the day of analysis. Since fmol amounts of internal standard were used, monitoring the recovery at this stage required LC/MS/MS analysis. Recovery of the internal standard from control DNA digested samples by LC/MS/MS was over 70%.

Calibration plot
A calibration curve, based on the area ratios of dG-C8-ABP (m/z 435 -> m/z 319) and d9-dG-C8-ABP (m/z 444 -> m/z 328) internal standard versus the concentration ratios, was generated from a series of samples containing a constant amount of the d9 internal standard (280 fmol) and varying amounts of dG-C8-ABP (0–230 fmol). This resulted in a linear plot with correlation coefficient of 0.999, a slope of 1.09 and an intercept of 0.0001 (Figure 1).


Figure 1
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  		Fig. 1 Calibration curve. This plot is based on the area ratios of dG-C8-ABP (m/z 435 -> m/z 319) and d9-dG-C8-ABP (m/z 444 -> m/z 328) internal standard versus the concentration ratios, and was generated from a series of samples containing a constant amount of the d9 internal standard (280 fmol) and varying amounts of dG-C8-ABP (0–230 fmol). Correlation coefficient = 0.999; slope = 1.09; intercept = 0.0001.

 
Liquid chromatography and mass spectrometry
After immunoaffinity recovery of the internal standard and dG-C8-ABP from the tissue DNA, further purification of the eluant was carried out with an automated online column switching system as described earlier (10). Briefly, this is a liquid handling system consisting of a manual injector (Rheodyne 2276, Rhonert Park, CA, USA), two automated switching valves (Rheodyne EV700–100), two binary HPLC pumps (Agilent 1100 Series), one trap column (POROS R2–10, 2.1 x 30 mm; Applied Biosystems, Foster City, CA, USA) and one analytical column (Eclipse XDB-C18 5 µm, 2.1 x 50 mm HPLC, Agilent Technologies, Wilmington, DE, USA). One of the pumps delivers a constant isocratic flow of 50% water and 50% methanol at a flow rate of 200 µl/min, to maintain the source conditions in the mass spectrometer; the other delivers a gradient to concentrate and wash the sample and then transfer it to the analytical column for analysis.

The samples were injected in 20 µl of methanol with the gradient pump delivering 100% water at 800 µl/min for 3 min, to load the sample onto the POROS column for concentration and desalting. The column was then washed for 3 min with 60% water and 40% methanol at a flow rate of 800 µl/min. At 6.1 min the valves were switched to reverse the flow through the POROS column onto the analytical column and into the mass spectrometer at 200 µl/min. This was followed by an elution gradient of 50/50 water/methanol to 99% methanol in 12 min and then to 100% water at 20 min. At 22 min, the system was switched to the starting configuration (100% water at 800 µl/min from the gradient pump through the POROS column to waste and 50:50 water:methanol at 200 µl/min from the isocratic pump through the analytical column and into the mass spectrometer).

Mass spectrometry
These experiments were carried out on an Applied Biosystems API 3000 tandem quadrupole mass spectrometer with a Turbo Ionspray Source operated in the positive ion mode. Detection was by multiple reaction monitoring using the transitions m/z 444 -> m/z 328 for the d9 internal standard and m/z 435 -> m/z 319 for the analyte (Figure 2) (10). The nebulizer gas was set at 9 psi, the curtain gas at 13 psi and the collision gas at 6 mtorr; the desolvating temperature was 335°C. The spray voltage was 5500; the declustering potential was 46 volts; focusing potential: 180 volts; entrance potential: –10 volts; collision energy: 35 volts and a collision cell exit potential of 20 volts. To avoid accumulation or carryover on the trap or analytical columns after a number of runs, and since we noticed that the recovery of the internal standard began to decrease after every five sample analyses, it was established that after four or five human DNA analyses, both trap and analytical columns were washed with 90% isopropanol for 30 min, followed by 1 h of 25:75 water/methanol and, finally, equilibration of the column to 50:50 water methanol for 30 min before the next run.


Figure 2
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  		Fig. 2 Collision-induced mass spectrum of the dG-C8-ABP adduct, demonstrating the loss of the deoxyribose to give the m/z 435 -> m/z 319 transition used for quantification by LC/MS/MS.

 
Hb adduct in human blood sample analysis
Blood samples were selected from those subjects whose bladder DNA contained detectable DNA 4-ABP adduct levels. Hb adduct detection assays were performed essentially as described previously (16). Briefly, solutions of Hb were prepared by lysis of red cells with distilled H2O and toluene, centrifugation, and dialysis of the supernatant against distilled H2O. After addition of an aliquot of internal standard, adducts were hydrolyzed by addition of NaOH to 0.1 M, followed by incubation at room temperature for 1 h. Amines were extracted with hexane, which was subsequently dried over MgSO4, and derivatized with (C2F5CO)2O and (CH3)3N. Hexane was removed using a rotary evaporator and samples were redissolved in 20 µl hexane for GC/MS analysis.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
LC/MS/MS optimization
The scheme for the method is outlined in Figure 3. Fresh internal standard was prepared and added after DNA extraction, at the first step of the digestion procedure. Amounts of deuterated standard added ranged from 56 to 282 fmol. Prior to analysis of DNA from human tissues, the optimized method was evaluated by application to control DNA samples isolated from TK6 lymphoblastoid cells, human placenta, rat liver DNA from 4-ABP-treated and untreated rats, and calf thymus DNA. The chromatogram in Figure 4 illustrates the complete digestion of 100 µg calf thymus DNA to its component deoxynucleosides.


Figure 3
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  		Fig. 3 Schematic flowchart for sample preparation and quantitative analysis of the dG-C8-ABP adduct.

 


Figure 4
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  		Fig. 4 LC chromatogram illustrating complete digestion of calf thymus DNA to its component deoxynucleosides with phosphodiesterase I Type II and alkaline phosphatase.

 
The mass spectrum of dG-C8-ABP (Figure 2) shows the [M+H+]+1 ion for the parent compound at m/z 435.1, which can generate a fragment ion at m/z 319.1 after the loss of the deoxyribose moiety (116 Da). As discussed previously (10), this fragmentation pattern allows the use of selected reaction monitoring (SRM) for confirmation of the chemical structure of the adduct. In this study, we followed SRM of both the in vivo adduct (m/z 435.1 -> m/z 319.1) and the internal deuterated standard (m/z 444.1 -> m/z 328.1) for characterization and quantification of dG-C8-ABP adducts. The limit of quantification was determined with the deuterated internal standard, d9-dG-C8-ABP, and found to be ~700 amol (300 femtograms), with a signal to noise ratio of 3. This would enable detection of at least 2 adducted bases in 109 bases. Correlations of the response to the amount of analyte and the purity of the internal standard were also examined. The calibration plot presented in Figure 1 represents the area ratio between dG-C8-ABP and d9-dG-C8-ABP. The resulting intercept of 0.0001 indicates the purity of the internal standard.

Application of the optimized SRM and column switching method to control DNA samples produced the data summarized in Table II. In each control sample, 100 µg DNA was hydrolyzed and spiked with a known amount of the deuterated internal standard, added at the beginning of the digestion step. Samples were then purified by immunoaffinity chromatography and analyzed by LC/MS. No dG-C8-ABP adduct was detected in TK6 cell DNA, human placental DNA or control rat liver DNA. On the other hand, liver DNA from rats treated with 4-ABP contained dG-C8-ABP at a level of 6 adducts in 107 bases (175 fmol). Contrary to expectations, commercial calf thymus DNA was also found to contain detectable levels of dG-C8-ABP adducts, even after potential artifactual contamination had been ruled out by appropriate experiments. Multiple batches of commercially available calf thymus DNA were shown to contain reproducible levels of the dG-C8-ABP adduct, at an average level of 1 adduct in 107 bases (30 fmol). Recovery of the internal standard from control samples averaged ~70%.


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  		Table II Levels of dG-C8-ABP detected in control DNA samples used in method validation

 
dG-C8-ABP adducts in control and human bladder tissues
To calculate the adduct amount in each sample, the integrated areas of the adduct peaks in the m/z 435.1 -> m/z 319.1 chromatograms were divided by the corresponding areas in the m/z 444.1 -> m/z 328.1 chromatograms for the internal standard. This ratio was multiplied by the known amount of deuterated internal standard added to the sample. The results, expressed in fmol, were then converted to adducts per nucleobase. SRM chromatograms for controls and human bladder samples are presented in Figure 5 showing the m/z 444.1 -> m/z 328.1 transition for the internal standard and the m/z 435.1 -> m/z 319.1 transitions for the samples. Analysis of liver DNA from untreated rats showed baseline levels of adduct, while that from treated animals resulted in the detection of 6 adducts in 107 bases (175 fmol). DNA from TK6 cells did not contain detectable levels of dG-C8-ABP adducts, while calf thymus DNA contained adduct levels of ~1 adduct in 107 nucleobases. Chromatograms of human DNA in Figure 5 show analyses of tumor and non-tumor DNA from the same bladder cancer patient. Analysis of non-tumor DNA resulted in detection of 23 adduct in 109 bases, whereas DNA from tumor contained ~36 adducts in 109 bases.


Figure 5
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  		Fig. 5 LC/MS/MS chromatograms from representative samples. Upper left: calf thymus DNA; upper right: DNA from TK6 cells; center left: liver DNA from untreated rats; center right: liver DNA from a 4-ABP-treated rat. Lower panels show analyses of non-tumor and tumor DNA from the same subject.

 
From the 27 subjects 44% of the samples (12 subjects) had quantifiable levels of dG-C8-ABP adducts in either tumor or non-tumor surrounding DNA. The range of detected adducts in the bladder DNA was from 5 to 80 adducts in 109 bases, corresponding to 3–48 fmol in 200 µg DNA. Table I presents the summary of demographic information and dG-C8-ABP adduct detected from the 27 analyzed subjects. From the 14 DNA pairs analyzed (tumor and non-tumor surrounding from the same subject), only one subject had detectable adducts in both tumor and non-tumor DNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
DNA adduct measurement has played an important role in assessing the impacts of environmental factors in the etiology of cancer, and provides a useful parameter for cancer risk assessment. Adduct levels serve as biomarkers of biologically effective dose, indicating the amount of carcinogen bound to DNA in target or surrogate tissues. Previous studies have estimated levels of DNA adduct thought to be necessary for tumor initiation to occur (17). However, this information was obtained mainly from studies in experimental animals exposed to chemical carcinogens and analysis of DNA isolated from induced tumors. In certain of these studies, exposure to 4-ABP resulted in a clear relationship between bladder tumor yield and DNA adduct concentration (18); the preferentially adducted sites on DNA were at the C8 position of deoxyguanosine and deoxyadenosine bases (19). Comparable studies in humans have been limited to date. One such investigation (8) reported detection of DNA adducts in human bladder biopsies by GC/MS, which strongly correlated with tumor grade, although the findings indicated no correlation with adduct levels in white blood cell DNA.

Previous investigations of 4-ABP DNA adducts in human tissues (pancreas, urinary bladder, placenta and lung) have employed various analytical methods, including 32P-post-labeling (11,20), immunoassays (21), and GC- and LC-mass spectrometry (8,22). In our study, we used an optimized LC/MS/MS method and were able to detect dG-C8-ABP adducts in urinary bladder DNA of 44% of subjects. The presence and level of DNA adducts in these subjects at the time of surgery presumably reflect recent exposure to 4-ABP and/or adducts that have escaped repair over some unknown period of time. The absence of quantifiable adducts in the remaining 56% of subjects does not necessarily rule out the possibility of exposure, since in two analyzed samples, adducts were detectable but not quantifiable inasmuch as the levels fell below the MS limit of quantification.

To our knowledge, there have been three studies of 4-ABP DNA adducts in human bladder tissue that included a significant epidemiological component. The first, described in several publications (11,20), used 32P-post-labeling for detection and quantification and found adducts at levels typically <1 per 108 nucleotides. There is a reasonable case that the analyte detected is indeed the dG-C8-ABP adduct, but it is recognized as not being conclusive. The second study (21) used immunohistochemical quantification and gave results in terms of mean relative staining intensity rather than adducts per nucleotide. Both studies found statistically significant effects of cigarette smoking on adduct levels as might be expected, but, given the relative lack of specificity of the assays used, it may be that the assays were responding to some undefined, smoking-related DNA damage other than 4-ABP adducts. The analytical method of the third study (8) was GC-MS, which is more specific than post-labeling or immunohistochemistry but still not without limitations because the mass spectrometry is performed with a single sector instrument. A complex relationship between smoking, tumor grade and diet was described. Again, smoking appeared to be a contributing factor to the levels of 4-ABP adducts detected. A notable aspect of this study is that mean adduct levels were nearly 1 per 106 nucleotides and ranged up to >1 in 105. The present study of 4-ABP adducts in human bladder DNA uses the most specific analytical methodology to date, although it is not the first to use tandem MS to quantify the dG-C8-ABP adduct in some human tissue: others have examined human pancreatic tissue (22). Both found adduct levels lower by 1–2 orders of magnitude than have been found using GC-MS (but perhaps higher than found using 32P-post-labeling), and both highlight extreme variability among individuals that does not reflect the relatively uniform exposure to 4-ABP apparent from the many reports of Hb adduct levels.

In previous studies, cigarette smoking appeared to be the primary source of exposure leading to formation of dG-C8-ABP adducts found in the urinary bladder and placenta (11,20,23). In our study, adducts cannot be associated with direct exposure to cigarette smoke exposure, since 26 out of 27 of our studied subjects were identified as non-smokers at the time of surgery. However, exposure to environmental tobacco smoke may have led to adduct formation. Other sources of exposure to 4-ABP are also known, such as the use of commercial hair dyes (4) which have been suggested to contribute to an increased risk of bladder cancer (24). Diet has also been identified as a source of 4-ABP. In animal studies, high levels of Hb adducts of 4-ABP have been reported in untreated animals from different laboratories fed various commercial standard diets (25).

In our study, tumor grade showed no correlation with DNA adduct levels. Since our subjects were identified as a non-smoking population, it is not possible to establish an association between smoking status and tumor grade as reported in a previous study involving analysis of bladder biopsy material (8). In that study, the presence of 4-ABP adducts was clearly associated with current smoking in patients with higher grade tumor, while in our study recent smoking was not known to occur. In another study (26) in which smoking and 4-ABP DNA adducts in human breast cancer were evaluated, smoking status was correlated with levels of 4-ABP DNA adducts in tissue adjacent to tumors, but not to adduct levels in tumor tissue.

Active cigarette smoking has a demonstrable effect on a person's exposure to 4-ABP. Typically, active smokers exhibit a several-fold elevation in Hb adduct levels as compared with non-smokers (27), and smokers who quit exhibit a decline to non-smoker levels within 1–2 months (28). Previous studies of 4-ABP DNA adducts in humans used smoking status as a surrogate for 4-ABP exposure. Since the present study was conducted with a non-smoker population, we elected to use Hb adducts as a measure of recent 4-ABP exposure. The level of Hb adduct in our studied subjects (median, 48; geometric mean 75 pg/g) was higher than previously observed (12) in bladder cancer cases (geometric mean = 33.5 for non-smokers at blood draw), but the values fall well within the range previously observed. The observation of high values in non-smokers is also in keeping with the earlier study, which found that 14 of the 18 highest values (>400 pg/g) were found in non-smokers.

The use of 4-ABP Hb adducts as a biomarker of exposure and internal dose of 4-ABP has previously been discussed (29). The results of the present study indicated no correlation between levels of dG-C8-ABP DNA adducts and 4-ABP Hb adducts, a conclusion that is readily apparent from inspection of the data given in Table I even without any formal statistical testing. A possible interpretation relies on the lack of evidence on the persistence of specific DNA adducts in the tissues in question. It is not possible to determine the time of formation of the observed adducts. In addition, activation or detoxification processes as well as DNA repair mechanisms may be tissue-specific and therefore correlation between Hb adducts and biopsies DNA adducts should not necessarily be expected. It has been noted (30) that when metabolism is complex and/or takes place in one or more compartments which are distant from the target, or when other physiological variables have a significant impact on adduct formation it becomes difficult to predict the relationship between exposure and adducts or adducts and effects. Gene polymorphisms may also play an important role in the formation of DNA and protein adducts (31). An epidemiological study of 67 smokers in which aromatic-DNA adduct levels were examined found high DNA adduct levels in slow acetylators for both NAT1 and NAT2. In contrast, no effect of genotype on 4-ABP Hb levels was observed (32).

When this study was initiated, the use of Hb adducts as a biomarker was presumed to provide a better measure of 4-ABP exposure than smoking status. Equally important, by using a non-smoker population, we expected to avoid confounding effects of smoking such as enzyme induction on the formation of DNA adducts. Nevertheless, we observed no evidence of an association between exposure as determined by Hb adduct levels and DNA adduct levels. A reasonable conclusion to be drawn from the results of this and previous studies is that biological variation in the dynamics that govern DNA adduct levels predominates over exposure variation in determining DNA adducts of 4-ABP in the bladder and that cigarette smoking has a pronounced effect on those dynamics.


   Acknowledgments
 
We thank Dr Misun Park and Mr J. Mellor for synthesis of standard dG-C8-ABP, Dr Fred A. Beland, NCTR, for the generous contribution of the d9-dG-C8-ABP internal standard, and Ms Yuhong Xiang for expert technical assistance. This work was supported by grants PO1-ES006052 and P30-ES002109 from the National Institute for Environmental Health Sciences, NIH.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Jung I. and Messing E. (2000) Molecular mechanisms and pathways in bladder cancer development and progression. Cancer Control 7:325–334.[Medline]
  2. Woo Y.T. and Lai D.Y. (2001) Aromatic amino and nitro-amino compounds and their halogenated derivatives. In Bingham E., Cohrssen B., Powell C.H. (Eds.). Patty's Toxicology(Wiley, New York) pp. 969–1105.
  3. Talaska G. (2003) Aromatic amines and human urinary bladder cancer: exposure sources and epidemiology. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 21:29–43.[CrossRef][ISI][Medline]
  4. Turesky R.J., Freeman J.P., Holland R.D., Nestorick D.M., Miller D.W., Ratnasinghe D.L., Kadlubar F.F. (2003) Identification of aminobiphenyl derivatives in commercial hair dyes. Chem. Res. Toxicol. 16:1162–1173.[CrossRef][ISI][Medline]
  5. Kadlubar F.F. (1994) DNA adducts of carcinogenic aromatic amines. In Hemminki K., Dupple A., Shuker D.E.G., Kadlubar F.F., Segerback D., Bartsch H. (Eds.). DNA Adducts Identification and biological significance pp. 199–216 IARC Scientific publications No. 125, IARC, Lyon.
  6. Feng Z., Hu W., Rom W.N., Beland F.A., Tang M.S. (2002) N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation. Biochemistry 41:6414–6421.[CrossRef][Medline]
  7. Feng Z., Hu W., Rom W.N., Beland F.A., Tang M.S. (2002) 4-Aminobiphenyl is a major etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human p53 gene. Carcinogenesis 23:1721–1727.[Abstract/Free Full Text]
  8. Airoldi L., Orsi F., Magagnotti C., Coda R., Randone D., Casetta G., Peluso M., Hautefeuille A., Malaveille C., Vineis P. (2002) Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies. Carcinogenesis 23:861–866.[Abstract/Free Full Text]
  9. Koc H. and Swenberg J.A. (2002) Applications of mass spectrometry for quantitation of DNA adducts. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 778:323–343.[ISI][Medline]
  10. Doerge D.R., Churchwell M.I., Marques M.M., Beland F.A. (1999) Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS. Carcinogenesis 20:1055–1061.[Abstract/Free Full Text]
  11. Talaska G., Schamer M., Skipper P.L., Tannenbaum S.R., Caporaso N., Unruh L., Kadlubar F.F., Bartsch H., Malaveille C., Vineis P. (1991) Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts and urinary mutagenicity. Cancer Epidemiol. Biomarkers Prev. 1:61–66.[Abstract]
  12. Skipper P.L., Tannenbaum S.R., Ross R.K., Yu M.C. (2003) Nonsmoking-related arylamine exposure and bladder cancer risk. Cancer Epidemiol. Biomarkers Prev. 12:503–507.[Abstract/Free Full Text]
  13. Kriek E. (1971) On the mechanism of action of carcinogenic aromatic amines. II. Binding of N-hydroxy-N-acetyl-4-aminobiphenyl to rat-liver nucleic acids in vitro. Chem. Biol. Interact. 3:19–28.[CrossRef][ISI][Medline]
  14. Lee M.-S. and King C.M. (1981) New synthesis of N-(guanosin-8-yl)-4-aminobiphenyl and its 5'-monophosphate. Chem. Biol. Interact. 34:239–248.[CrossRef][ISI][Medline]
  15. Groopman J.D., Skipper P.L., Donahue P.R., Trudel L.J., Wildschutte M., Kadlubar F.F., Tannenbaum S.R. (1992) Monoclonal antibodies and rabbit antisera recognizing 4-aminobiphenyl-DNA adducts and application to immunoaffinity chromatography. Carcinogenesis 13:917–922.[Abstract/Free Full Text]
  16. Skipper P.L. and Stillwell W.G. (1994) Aromatic amine-hemoglobin adducts. In Everse J., Vandegriff K.D., Winslow R.M. (Eds.). Methods in Enzymology(Academic Press, San Diego) Vol. 231: pp. 643–649.[ISI][Medline]
  17. Otteneder M. and Lutz W.K. (1999) Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts. Mutat. Res. 424:237–247.[ISI][Medline]
  18. Beland F.A., Fullerton N.F., Smith B.A., Poirier M.C. (1992) DNA adduct formation and aromatic amine tumorigenesis. Prog. Clin. Biol. Res. 374:79–92.[Medline]
  19. 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: Carcinogenesis and Mutagenesis(Springer-Verlag, Heidelberg) Vol. 94. pt I. pp. 267–325.
  20. Talaska G., Al-Juburn A.Z., Kadlubar F.F. (1991b) Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct. Proc. Natl Acad. Sci. USA 88:5350–5354.[Abstract/Free Full Text]
  21. Curigliano G., Zhang Y.J., Wang L.Y., Flamini G., Alcini A., Ratto C., Giustacchini M., Alcini E., Cittadini A., Santella R.M. (1996) Immunohistochemical quantitation of 4-aminobiphenyl-DNA adducts and p53 nuclear overexpression in T1 bladder cancer of smokers and nonsmokers. Carcinogenesis 17:911–916.[Abstract/Free Full Text]
  22. Ricicki E.M., Soglia J.R., Teitel C., Kane R., Kadlubar F.F, Vouros P. (2005) Detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human pancreas tissue using capillary liquid chromatography-microelectrospray mass spectrometry. Chem. Res. Toxicol. 18:692–699.[CrossRef][ISI][Medline]
  23. Kadlubar F.F., Talaska G., Lang N.P., Benson R.W., Roberts D.W. (1988b) Assessment of exposure and susceptibility to aromatic amines carcinogens. In Bartsch H., Hemminki K., O'Neill I.K. (Eds.). Methods for Detecting DNA Damaging Agents in Humans: Applications in Human Epidemiology and Prevention(IARC Scientific Publications No. 89, IARC, Lyon) pp. 166–174.
  24. Gago-Dominguez M., Bell D.A., Watson M.A., Yuan J.M., Castelao J.E., Hein D.W., Chan K.K., Coetzee G.A., Ross R.K., Yu M.C. (2003) Permanent hair dyes and bladder cancer: risk modification by cytochrome P4501A2 and N-acetyltransferases 1 and 2. Carcinogenesis 24:483–489.[Abstract/Free Full Text]
  25. Richter E., Rosler S., Becker A. (2000) Effect of diet on hemoglobin adducts from 4-aminobiphenyl in rats. Arch. Toxicol. 74:203–206.[CrossRef][ISI][Medline]
  26. Faraglia B., Chen S.Y., Gammon M.D., et al. (2003) Evaluation of 4-aminobiphenyl-DNA adducts in human breast cancer: the influence of tobacco smoke. Carcinogenesis 24:719–725.[Abstract/Free Full Text]
  27. Bryant M.S., Skipper P.L., Tannenbaum S.R., Maclure M. (1987) Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers. Cancer Res. 47:602–608.[Abstract/Free Full Text]
  28. Maclure M., Bryant M.S., Skipper P.L., Tannenbaum S.R. (1990) Decline of the hemoglobin adduct of 4-aminobiphenyl during withdrawal from smoking. Cancer Res. 50:181–184.[Abstract/Free Full Text]
  29. Skipper P.L. and Tannenbaum S.R. (1994) Molecular dosimetry of aromatic amines in human populations. Environ. Health Perspect. 102:Suppl. 6, 177–121.
  30. Skipper P.L. and Tannenbaum S.R. (1990) The role of protein adducts in the study of chemical carcinogenesis. Mutation and the Environment, Part C(Wiley-Liss, Inc., New York) pp. 301–310.
  31. Vineis P. (1992) The use of biomarkers in epidemiology: the example of bladder cancer. Toxicol. Lett. 64/65:463–467.[CrossRef]
  32. Godschalk R.W., Dallinga J.W., Wikman H., Risch A., Kleinjans J.C., Bartsch H., Van Schooten F.J. (2001) Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1,GSTT1, NAT1 and NAT2. Pharmacogenetics 11:389–398.[CrossRef][ISI][Medline]
Received April 12, 2006; revised July 7, 2006; accepted August 1, 2006.


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