MS Following In Vitro

Chem. Res. Toxicol. 2003, 16, 1251-1263

1251

Identification of DNA Adducts Using HPLC/MS/MS Following In Vitro and In Vivo Experiments with Arylamines and Nitroarenes Christopher R. Jones†,‡ and Gabriele Sabbioni*,‡,§ Department of Environmental & Occupational Medicine, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, England, Walther-Straub-Institut fu¨ r Pharmakologie und Toxikologie, Ludwig-Maximilians-Universita¨ t Mu¨ nchen, D-80336 Mu¨ nchen, Germany, and Institute of Environmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland Received July 8, 2002

Arylamines and nitroarenes are suspected of playing a key role in chemical carcinogenesis. Therefore, the study of DNA adduct formation is an important step to determine the genotoxic potential of these compounds. Calf thymus DNA was modified in vitro by reaction with activated N-hydroxyarylamines: 2-chloroaniline (2CA), 4-chloroaniline (4CA), 2-methylaniline (2MA), 4-methylaniline (4MA), 2,4-dimethylaniline (24DMA), 2,6-dimethylaniline (26DMA), 2-aminobiphenyl (2ABP), 3-aminobiphenyl (3ABP), and 4-aminobiphenyl (4ABP). Female Wistar rats (n ) 2) were given a single dose of the above arylamines and their analogous nitro derivatives by oral gavage and sacrificed after 24 h. Hepatic DNA and in vitro modified DNA were hydrolyzed enzymatically to individual 2′-deoxyribonucleosides. Adducts were determined using HPLC/MS/MS by comparison to synthesized standards. The hydrolysis efficiency was monitored by HPLC with UV detection. Each arylamine described above formed adducts to 2′-deoxyguanosine and 2′-deoxyadenosine after in vitro reaction with DNA. DNA adducts were found in rats dosed with 4ABP or with 4-nitrobiphenyl. DNA adducts were not detected in rats dosed with 2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, 2-chloronitrobenzene, 4-chloronitrobenzene, 2-nitrotoluene, and 4-nitrotoluene. All compounds formed hydrolyzable hemoglobin adducts. Therefore, biologically available N-hydroxyarylamines yielded hemoglobin adducts but not hepatic DNA adducts, except for 4ABP.

Introduction Arylamines and nitroarenes are very important intermediates. N-Oxidation is a key step in the metabolism of arylamines to toxic products. Arylamines are metabolized to highly reactive N-hydroxyarylamines (1) by CYP.1 N-Hydroxyarylamines can be further metabolized to sulfonyloxy-arylamines, N-acetoxyarylamines, or N-hydroxyarylamine-N-glucuronides. These highly reactive intermediates are responsible for the genotoxic effects of this class of compounds (2, 3). DNA adducts of arylamines have been found in several organs of exposed experimental animals (2, 4). 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 (5). The levels of DNA adduction are typically in the range of 1 in 106 to 1 in 109 normal nucleotides (6). Therefore, highly sensitive techniques are required for the analysis of small amounts of DNA (1300 µg), which are available in human studies. The various methods used for DNA adduct determinations have been recently reviewed (7). DNA adducts of aryl* To whom correspondence should be addressed. E-mail: [email protected]. † University of Newcastle upon Tyne. ‡ Ludwig-Maximilians-Universita ¨ t Mu¨nchen. § Institute of Environmental and Occupational Toxicology.

amines have been determined with radiolabeled compounds, with 32P-postlabeling (8), with immunoassays (9), with HPLC and electrochemical detection (10), and with HPLC/MS (11-14). Immunoassays have been used for the determination of DNA adducts deriving from 4ABP (9). In recent years, HPLC/MS/MS has assumed an important role in bioanalytical chemistry in terms of structure characterization, trace level detection, and 1 Abbreviations: A, adenine; 2ABP, 2-aminobiphenyl; 3ABP, 3-aminobiphenyl; 4ABP, 4-aminobiphenyl; AF, ammonium formate; AP, alkaline phosphatase; C, cytosine; 2CA, 2-chloroaniline; 4CA, 4-chloroaniline; CE, collisional energy; CID, collision-induced dissociation; CYP, cytochrome P450 monooxygenases; ct-DNA, calf thymus DNA; DAD, diode array detector; dC, 2′-deoxycytidine monohydrochloride; dG, 2′-deoxyguanosine monohydrate; dG-C8-4ABP, N-(deoxyguanosine8-yl)-4ABP; 24DMA, 2,4-dimethylaniline; 26DMA, 2,6-dimethylaniline; dG-C8-2CA, N-(2′-deoxyguanosine-8-yl)-2-chloroaniline; dG-C8-4CA, N-(2′-deoxyguanosine-8-yl)-4-chloroaniline; dG-C8-24DMA, N-(2′-deoxyguanosine-8-yl)-2,4-dimethylaniline; dG-C8-26DMA, N-(2′-deoxyguanosine-8-yl)-2,6-dimethylaniline; dG-C8-2MA, N-(2′-deoxyguanosine8-yl)-2-methylaniline; dG-C8-4MA, N-(2′-deoxyguanosine-8-yl)-4-methylaniline; dN, 2′-deoxyribonucleoside; DNase I, deoxyribonuclease I; dT, thymidine; EI, electron impact ionization; ESI, electrospray ionization; G, guanine; 2MA, 2-methylaniline; 4MA, 4-methylaniline; MgCl2, magnesium chloride hexahydrate; NaOAc, sodium acetate; NEt3, triethylamine; NP1, nuclease P1; 5′P-dA, 2′-deoxyadenosine-5′monophosphate; 5′P-dC, 2′-deoxycytidine-5′-monophospate; 5′P-dG, 2′-deoxyguanosine-5′-monophosphate; PFPA, pentafluoropropionic anhydride; rA, adenosine; rC, cytidine; rG, guanosine; RAL, relative adduct level; RSD, relative SD () SD/mean × 100%); rU, uridine; SDS, sodium dodecyl sulphate; sodium citrate, trisodium citrate dihydrate; SVPD, snake venom phosphodiesterase I; T, thymine; 245TMA, 2,4,5trimethylaniline; Tris, tris(hydroxymethyl)aminomethane; ZnCl2, zinc chloride; ZnSO4, zinc sulfate.

10.1021/tx020064i CCC: $25.00 © 2003 American Chemical Society Published on Web 09/11/2003

1252 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

quantitation. The major advantages of HPLC/MS/MS are (i) selectivity and specificity, (ii) additional evidence from characteristic retention times, and (iii) the potential for quantitative analyses without chemical derivatization reactions. The major drawback of HPLC/MS/MS analyses with ESI is ion suppression effects of, in part, unknown mechanisms (15, 16). This effect limits the amount of sample used for analyses (14). In view of these aforementioned features, a number of laboratories have been interested in replacing the 32P-postlabeling methods with the new HPLC/MS/MS technique (11-13, 17). HPLC/MS analyses were successfully conducted on the dG-C8 adduct of 4ABP in rodents. Doerge et al. (12) developed a quantitative isotope dilution method for analysis of dG-C8-4ABP in mice. Column switching valves were used to perform on-line sample concentration and cleanup. The trapped sample was then analyzed by HPLC/MS/MS. The determination limit for a sample containing 100 µg of DNA was 0.7 dG-C8-4ABP adducts in 107 normal nucleotides. The arylamines and nitroarenes investigated in the present study are used to produce pesticides, dyes, antioxidants, pharmaceuticals, and other products (18). Humans are exposed, for example, to 2MA (24) and 4CA (19) at the workplace, to 26DMA from the pharmaceutical lidocaine (20), and to methylanilines and dimethylanilines from cigarette smoke (21, 22). 2MA, 26DMA, and 4CA are classified in group 2B as possibly carcinogenic in humans (23). The risk of chronic exposure to these compounds in the occupational setting may have been underestimated. For example, 2MA has been associated with increased risk of bladder cancer in exposed industrial rubber workers (24). From experimental models, it is considered likely that DNA adduct formation in humans should provide a valid marker of molecular dosimetry and be suggestive of increased human cancer risk (5). Therefore, methods of detecting DNA adducts of arylamines in vivo are required for assessment of their genotoxic potential. We were interested in developing a HPLC/MS/MS method to quantitate DNA adducts formed in vitro and in vivo. Our goal was to develop a quantitative assay for the analysis of potential DNA adducts formed in rats, following administration of a selection of structurally related nitroarenes and arylamine analogues. The chemicals of this study, except for 2ABP and 3ABP, have previously been shown to form hemoglobin (Hb) adducts in rats (25-30). Such adducts result from biologically available N-hydroxyarylamines, which oxidize to nitrosoarenes in the erythrocytes and then react with Cys to form sulfin amide adducts with Hb (31, 32). One point of interest was to ascertain whether the same arylamines would form DNA adducts at sufficiently high levels to facilitate detection by HPLC-ESI-MS/MS. To date, the identification and quantitation of DNA adducts, in vivo, for this class of compound, except 4ABP, have been limited to analysis by 32P-postlabeling, due to the low levels of damaged DNA residues as compared to normal DNA bases.

Materials and Methods Chemicals. 1-Butanol (spectrophotometric grade, 99+ %) was obtained from Acros (Geel, Belgium). ct-DNA sodium salt (95%), dC, dT, 5′P-dG, and ethanol (spectrophotometric grade) were obtained from Aldrich (Taufkirchen, Germany). NP1 from

Jones and Sabbioni Penicillium citrinum, Rnase T1 from Aspergillus oryzae, Rnase A from bovine pancreas, and Proteinase K from Tritirachium album were obtained from Roche Diagnostics (Mannheim, Germany). dG, formic acid puriss. p.a., MgCl2 (Microselect grade), ZnSO4 puriss. p.a., ZnCl2 (Microselect grade), NaOAc, NEt3 puriss. p.a., pyruvonitrile, AF (Microselect grade), dimethyl sulfoxide (DMSO) (Microselect grade), and Tris (Microselect grade) were obtained from Fluka (Deisenhofen, Germany). Tritriplex EDTA p.a. and SDS salt, >99%, were obtained from Merck (Darmstadt, Germany). rA, rC, rG, rU, C, A, T, and G were obtained from Serva (Heidelberg, Germany). 2′-Deoxyadenosine (dA), SVPD type II from Crotalux adamentous (catalog no. P6877), DNase I type II from bovine pancreas (catalog no. D4527), AP type III from Escherichia coli (catalog no. P4252), 5′P-dC, and 5′P-dA were obtained from Sigma (Taufkirchen, Germany). Acetonitrile (Baker ultragradient HPLC grade), methanol (Baker ultraresianalyzed), and water (Baker ultraresianalyzed) were obtained from Baker (Griesheim, Germany). dG-C8-2CA, dG-C8-4CA, dG-C8-2MA, dG-C8-4MA, dGC8-24DMA, and dG-C8-26DMA were synthesized according to Beyerbach et al. (34). All of the N-hydroxyarylamines (2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, and 4ABP) were synthesized from their corresponding nitro compounds according to refs 33 and 34. LiChrosolv HPLC grade water (H2O) was obtained from Merck. 2MA-d4, 4MA-d4, 24DMA-d3, 26DMAd3, and 4ABP-d9 were synthesized according to Sabbioni and Beyerbach (35). MS. Helium and nitrogen of 99.999% purity were used (Linde, Munich, Germany). An ion trap mass spectrometer (LCQ-Duo, Thermo Finnigan, San Jose, CA) was used in the positive ion mode, employing ESI. The instrument parameters were applied as follows: capillary temperature, 220 °C; nitrogen sheath gas flow, 80 (arbitrary units); auxiliary gas flow, 0 (arbitary units); and spray voltage, 5 kV. The remaining parameters were optimized by the autotune program to achieve maximum transmission of the [M + H]+ ion of dG-C8-4CA, using 100 pg/µL of dG-C8-4CA, at a flow rate of 250 µL/min; the heated capillary temperature was set at 220 °C. Structural information was obtained by further fragmentation using MS/MS. MS/MS spectra were obtained by ion trap CID. A CE was selected that optimized fragmentation of the [M + H]+ parent ion to the daughter ion [BH2]+. Treatment of Rats with Arylamines and Nitroarenes. Female Wistar rats (200-225 g) were obtained from the Zentralinstitut fu¨r Versuchstierzucht (Hannover, Germany). The test compounds were administered as 0.5 M solutions (0.1 mL per 100 g body weight) in 1,2-propanediol (arylamines) or tricaprylin (nitroarenes) by gavage to groups of two animals. After 24 h, the animals were anesthetized with ether, and blood (4-6 mL) was drawn into EDTA tubes by cardiac puncture. The livers were removed, washed with saline solution, then frozen in liquid nitrogen, and stored at -20 °C. To confirm the negative results, all animal experiments with nondeuterated arylamines (except for 4ABP) and all experiments with nitroarenes (except for 4NBP) were repeated with female Wistar rats at the Comparative Biology Center of the University of Newcastle, Newcastle upon Tyne, England. Two control rats were given only the solvent. Isolation of Erythrocytes from Whole Blood. Freshly drawn heparinized blood was centrifuged for 10 min to separate the erythrocytes from the plasma phase. The supernatant, containing the plasma, was removed and stored at -20 °C. The erythrocytes were washed three times with equal volumes of saline solution (0.9% NaCl). The erythrocytes were stored at -20 °C until isolation of Hb. Isolation and Purification of Hb. Isolation and purification of Hb were performed according to refs 26 and 35. An aliquot (2 mL) of thawed erythrocytes was added to ice cold H2O (8 mL) and maintained at 4 °C for 20 min, to facilitate lysis of cellular membranes. The membranes were removed by centrifugation. The supernatant was decanted carefully to a new glass tube, and Hb was precipitated with 4 vol of -20 °C EtOH. The

Identification of DNA Adducts Using HPLC/MS/MS precipitate was washed with EtOH-H2O (8:2), twice with EtOH, then once with EtOH-diethyl ether (3:1), and finally with diethyl ether. The Hb was dried in a desiccator over silica gel until a constant weight was attained. The Hb was stored at -20 °C until analysis. Determination of Hb Adducts. To Hb (40 mg) dissolved in 0.1 M NaOH and 0.01% SDS, the corresponding internal standard was added as follows: 2MA-d4 (1 µg) for 2MA, 4MA-d4, (1 µg) for 4MA, 24DMA-d3 (1 µg) for 24DMA, 4-bromoaniline (10 µg) for 4CA, 4ABP-d9 (200 ng, 1 µg, 10 µg) for 2ABP, 3ABP, and 4ABP, respectively. After it was gently shaken for 1 h at room temperature, the hydrolysate was extracted with hexane (6 mL). 245TMA (10 µg) was added as a recovery standard to the extracts of the 4ABP and 4CA experiment. The extracts of the 2MA, 4MA, 24DMA, 2ABP, and 3ABP experiment were evaporated to 1 mL, and 245TMA (1 µg) was added. The analyses were performed on a Hewlett-Packard chromatograph (HP 5890II) equipped with an autosampler (HP 7673) and interfaced to a mass spectrometer (HP 5989A). The PFPA derivatives of the aromatic amines were analyzed by splitless injection on to a fused silica capillary column (J + W; DB 1701; i.d. 0.25 mm; length 15 m, 1 µm film thickness) with a 1 m × 0.25 mm methyl-silyl retention gap (Analyt; Mu¨llheim, Germany). The injector and transfer line temperature were set at 180 °C. Helium was used as carrier gas with a flow rate of 1.5 mL/min. In the EI mode, the electron energy was 70 eV and the ion source temperature was 200 °C. The GC oven temperature was held at 50 °C for 1 min and then increased at 50 °C/ min to 160 °C. In the single-ion monitoring mode, the positive ions m/z ) 106 and 107 were monitored for 2MA (4MA), m/z ) 110 and 111 for 2MA-d4 (4MA-d4), and m/z ) 106, 120, 121 for 24DMA, m/z ) 123, 124 for 24DMA-d3, and m/z ) 135 for 245TMA. Each of these ions was detected with a dwell time of 50 ms. Helium was used as the carrier gas with a flow rate of 1.5 mL/min. The samples were quantified against a calibration curve obtained with standard solutions in hexane, for example, 2MA-d4 (1 µg), 245TMA (1 µg), and 0-100 ng of 2MA. 2MA, 4MA, 24DMA, and 245TMA eluted with a retention time of 3.70, 3.70, 4.19, and 5.20 min, respectively. 2ABP (tR ) 6.03 min, m/z ) 168, 169), 3ABP (tR ) 6.97 min, m/z ) 168, 169), 4ABP (tR ) 7.02 min, m/z ) 168, 169), 4ABP-d9 (tR ) 7.00 min, m/z ) 178), 4CA (tR ) 4.3 min, m/z ) 127, 129), 245TMA (tR ) 4.5 min, m/z ) 135), and 4BrA (tR ) 5.3 min, m/z ) 179, 181) were analyzed on the same column as described above but with a different temperature program: 1 min at 50 °C, 50 °C/min to 200 °C, 1 min at 200 °C, and 50 °C/min to 240 °C. For structural confirmation, the hexane extracts were dried over Na2SO4 and derivatized with PFPA as described in ref 35. The derivatives were blown to dryness with a gentle stream of nitrogen, taken up in 15 µL of hexane, and analyzed on the same column. In this case, the initial oven temperature, the injector temperature, and the transfer line temperature were set at 50, 200, and 200 °C, respectively. The oven temperature was increased at a rate of 50 °C/min to 200 °C, held for 1.2 min, and then heated at 50 °C/min to 240 °C and held for 3.2 min. The samples were analyzed with the MS in the EI or negative chemical ionization mode. All samples were extracted with hexane at neutral pH (3 mL of 0.1 M phosphate buffer, pH 7.4, containing 0.2% SDS () neutral hydrolysis) and worked up as described above to establish whether the arylamines detected were covalently bound. Isolation of DNA from Rat Liver. DNA was isolated and purified from rat liver essentially by the procedure described in the literature (36, 37). The DNA was redissolved in water at approximately 2 mg/mL and stored at -20 °C. Determination of DNA Concentration. The concentration and purity of extracted DNA samples were determined by UV spectrometry. Dilutions (1/50) of thawed DNA solutions were made in H2O and pipetted into quarz cuvettes. The absorbance was read between 220 and 300 nm wavelength. The DNA concentration was calculated at absorbance A260 assuming

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1253 50 µg/mL ) 1.0 absorbance unit at 260 nm. The UV absorbance was acquired between 220 and 300 nm to determine UV spectral ratios of A260/280 and A230/260. The concentration of DNA isolated from liver samples (3 g) was 1.9 ( 0.82 mg/g liver. The spectral ratios determined for DNA isolated from liver samples were A230/260 ) 0.46 ( 0.05 and A260/280 ) 1.86 ( 0.18 and were close to the theoretical values (A260/280 ) 1.8 and A230/260 ) 0.45 (41)). The maximum UV absorption was found at approximately 260 nm. Synthesis of 2′-Deoxyguanosine (dG) and dA Adducts. The dG and dA arylamine adducts were synthesized with slight modifications according to the procedure described in ref 34. Equivalent molar ratios of N-hydroxyarylamines (60 µmol) (2CA, 4CA; 2MA, 4MA; 24DMA, 26DMA; 2ABP, 3ABP, 4ABP) and NEt3 (8.4 µL, 60 µmol) were dissolved in THF (3 mL) at -45 °C in glass screw-capped tubes (GL18, Schott Glass, Mainz, Germany, 100 mm × 16 mm) with Teflon liners. An equivalent of pyruvonitrile (4.3 µL, 60 µmol) was added to yield the N-acetoxyarylamine, which was not isolated. The reaction was monitored by TLC (98:2, CHCl3:MeOH) and found to be complete after 90 min. Then, dA (15 mg, 52 µmol) or dG (15 mg, 52 µmol) in H2O (1 mL) and NEt3 (3.4 µL, 24 µmol) were added and incubated in a shaking bath overnight at 37 °C. The reaction mixture was evaporated, and the residue was reconstituted in H2O (5 mL). The aqueous phase was washed with diethyl ether (6 × 3 mL) and with EtOAc (3 × 3 mL). The EtOAc extracts were combined and concentrated in vacuo. The residue was reconstituted in MeOH and purified by preparative TLC (1:1 CHCl3:EtOH). Each discrete band of silica on the TLC plate was removed and extracted with MeOH (500 µL). HPLC/MS/MS was used to identify the zone containing the product from characteristic fragments, [M + H]+ and [BH2]+, consistent with formation of a covalent bond between dA (dG) and the arylamine and from cleavage of the glycosidic bond, respectively. In Vitro Reaction of ct-DNA with N-Acetoxyarylamines. The N-acetoxyarylamine solutions (-45 °C) (of 2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, and 4ABP (see above)) were added dropwise to a solution of ct-DNA (10 mg/1 mL H2O) in EtOH (1.75 mL), CHCl3 (0.75 mL), and NEt3 (3.4 µL) and then incubated in a shaking bath at 37 °C overnight. The mixture was washed six times with equivalent volumes of diethyl ether and EtOAc. The removal of unreacted N-hydroxyarylamine was confirmed by TLC analysis (98:2, CHCl3: EtOH). The DNA was precipitated with 5.0 M NaCl (1.5 mL) and EtOH (15 mL) at -20 °C overnight. The DNA was collected by centrifugation (1000g), washed with 70% EtOH (1 mL), and then redissolved at approximately 2 mg/mL in H2O. The concentration of DNA isolated from the in vitro modification reactions was 3.6 ( 1.15 mg/reaction. The spectral ratios determined for DNA isolated from ct-DNA from the in vitro modification reactions were A230/260 ) 0.47 ( 0.07 and A260/280 ) 1.76 ( 0.11 and were close to the theoretical values. Optimization of DNA Hydrolysis Conditions. Experiments were carried out to evaluate the efficiency of DNA digestion by determination of the dNs and of the adducts released from DNA (500 µg) using different combinations of enzymes. DNA samples (125 µg) from rats treated with 4ABP were spiked with ct-DNA (375 µg) to give DNA solutions of 500 µg. The DNA solution was pipetted into a 1.5 mL plastic eppendorf and spiked with dG-C8-4CA (100 pg) as internal standard. DNA was hydrolyzed with either two enzymes (nuclease P1 (NP1) and alkaline phosphatase type III (AP)); three enzymes (deoxyribonuclease I (DNase I), NP1, and AP); or four enzymes (DNase I, NP1, and snake venom phosphodiesterase I (SVPD) with AP). The samples were transferred to teflon tubes, extracted with EtOAc, reduced in vacuo, and reconstituted in H2O containing dG-C8-26DMA (100 pg). The samples were analyzed by HPLC/MS/MS. The response factor for dG-C8-4ABP was calculated from the integrated peak area of dG-C8-4ABP relative to recovery standard dG-C8-26DMA. The response factor was indicative of the digestion efficiency of the reaction.


Jones and Sabbioni

Table 1. Spectrum of Adducts Formed Following Reaction of dG, dA, or Whole ct-DNA with a Series of N-Acetoxyarylaminesa reaction with single base activated arylamine

adduct TLC (Rf)

HPLC tR (min)

2CA dG

13.8b

dA (0.35)

12.1d, 14.4d, 15.9d

dG

16.8b

dA (0.36)

18.6d

dG

14.1b

dA (0.19)

13.3d, 14.9d, 16.6d

dG

16.6b

dA (0.34)

18.6d

dG

16.2b

dA(0.53)

13.3d, 14.3d, 16.8d, 18.5d

dG

14.2b

dA (0.30)

14.4d

dG (0.11)

17.8d

dA (0.27)

18.4d

dG (0.16)

14.3d, 19.2d

dA (0.22)

14.1d

dG (0.47)

20.1c

4CA

2MA

4MA

24DMA

26DMA

2ABP

3ABP

4ABP

reaction with ct-DNA adduct dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA dC dG dT dA

tR (min) 10.8e

internal standard added before DNA hydrolysis standard

tR (min)

dG-C8-4ABP

19.9

dG-C8-4ABP

19.9

dG-C8-4MA

16.6

dG-C8-2MA

14.8

dG-C8-4ABP

20.3

dG-C8-4ABP

20.3

dG-C8-26DMA

14.2

18.5d 17.2e 14.0d, 19.2d

dG-C8-26DMA

13.5

14.1d 19.1e 20.1c

dG-C8-4CA

17.2

13.8b, 14.5e 18.5e 12.1d, 14.4d, 15.9d 12.6e 16.5b 13.4e, 15.2e, 18.3e 15.7,e 18.3d 14.4e 12.5e, 14.1b, 15.6e 14.2e 13.3d, 14.9d, 16.6d 15.4e 16.6b, 17.8e 13.5e, 18.6d 16.1b 14.2d, 16.7d, 18.4d 11.2e 13.8e, 14.3b, 16.1e 19.1e 12.6e, 14.4d 15.8e 17.7d

19.7e, 21.5e

a The dG and dA adducts were analyzed by HPLC-ESI-MS/MS. The [M + H]+ and [BH ]+ ions of each DNA adduct were acquired. 2 The adduct has been structurally characterized as the dG-C8-arylamine by NMR and MS (34). c The adduct has been structurally d characterized by MS and UV spectroscopy according to the literature. The structure of the synthetic equivalent has not been fully elucidated. e No synthetic equivalent has been synthesized.

b

For the two enzyme, three enzyme, and four enzyme digestion, response factors of 0.61, 1.44, and 1.70 were obtained. 1. Two Enzyme Hydrolysis of DNA. The DNA was sheered by sonication (3 min), in buffer (30 mM NaOAc (pH 5.3)/3 mM ZnSO4), in a total volume of 370 µL. NP1 (30 µL, 9 units) was added, and the reaction was incubated for 3 h in a shaking water bath at 37 °C. Then, 50 µL of buffer (500 mM Tris-HCl, pH 8.8) was added with 15 µL of AP (3 units). The reaction was incubated overnight at 37 °C. 2. Three Enzyme Hydrolysis of DNA. The DNA was sheered by sonication (3 min) in buffer (5 mM Bis-Tris (pH 7.1)/5 mM MgCl2) in a total volume of 350 µL. DNase I (50 µL, 250 units) was added, and the reaction was incubated for 3 h, in a shaking water bath, at 37 °C. A second buffer (50 µL, 300 mM NaOAc (pH 5.3)/3 mM ZnSO4) and 30 µL of NP1 (9 units) were added, and the reaction was incubated for a further 3 h at 37 °C. Finally, 60 µL of buffer (500 mM Tris-HCl, pH 8.8) and 15 µL of AP (3 units) were added, and the reaction was incubated overnight at 37 °C. 3. Four Enzyme Hydrolysis of DNA. The DNA was sheered by sonication (3 min) in buffer (5 mM Bis-Tris (pH 7.1)/5 mM MgCl2) in a total volume of 350 µL. DNase I (50 µL, 250 units) was added, and the reaction was incubated for 3 h in a shaking water bath at 37 °C. A second buffer (50 µL, 300 mM NaOAc (pH 5.3)/3 mM ZnSO4) and 30 µL of NP1 (9 units) were added, and the reaction was incubated for a further 3 h at 37 °C. Finally, 60 µL of buffer (500 mM Tris-HCl, pH 8.8), 40 µL of SVPD (0.026 units), and 15 µL of AP (3 units) were added and incubated overnight at 37 °C.

HPLC/DAD Determination of DNA Hydrolysis. Complete digestion of DNA was confirmed by analysis of dN content. An aliquot of the DNA hydrolysate (10 µL) was injected manually onto an HPLC system (Hewlett-Packard series 1100) coupled to a DAD. The dNs were separated from 5′P-dN, ribonucleosides, and DNA bases using a LiChrospher RP-18 column (250 mm × 4 mm, 5 µm) with a RP18 precolumn (4 mm × 4 mm) at 1 mL/ min in mobile phase (94:3:3, 10 mM AF:CH3CN:H2O) and detected at 265 nm wavelength. Under these conditions, the dNs eluted independent of each other and separately from the other nucleic acid constituents; retention times: 5′P-dC ) 1.6, 5′PdG ) 2.5, C ) 2.7, rC ) 3.2, 5P′-dA ) 3.3, G ) 3.7, rG ) 3.8, dC ) 4.4, T ) 5.1, A ) 6.1, rU ) 7.3, dG ) 10.1, dT ) 11.9, rA ) 16.3, dA ) 17.8 min. For determination of digestion efficiency, the peak areas of each dN were integrated and compared to a calibration line of known standards. The calibration line was made from aqueous solutions of combined dN standards, dC, dG, dT, and dA (0, 100, 500, and 1000 ng/10 µL), which were analyzed under identical chromatographic conditions. The precision of the assay was determined from replicate injections of the dN standards. The same solutions (0, 100, 500, and 1000 ng/10 µL) were injected 15 times over a period of 4 months. The RSD (RSD ) SD/mean × 100%) associated with the integrated peak areas increased as the amount of dN injected decreased. The RSD for each dN ranged between 3.7 and 5.7% showing that the assay was highly reproducible over an extended time period. When the precision of the method was determined from the biological matrix of interest, by injection of replicate aliquots of a single ct-DNA hydrolysate, the RSD

Identification of DNA Adducts Using HPLC/MS/MS

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1255

Table 2. Quantitation of dG-C8-Arylamine Adducts Released in Digested In Vitro Modified DNAa in vitro modified DNAb nonmodified control DNA 2CA 4CA 2MA 4MA 24DMA 26DMA 4ABP

internal standard

recovery standard

calibration linec

dG-C8 adducts (pmol/500 µg) 0

dG-C8-4ABP dG-C8-4ABP dG-C8-4MA dG-C8-2MA dG-C8-4ABP dG-C8-4ABP dG-C8-4CA

dG-C8-26DMA dG-C8-26DMA dG-C8-4CA dG-C8-4CA dG-C8-4CA dG-C8-4CA dG-C8-4MA

y ) 0.0117x + 0.0092 y ) 0.0034x + 0.0038 y ) 0.0045x + 0.0055 y ) 0.0030x - 0.0062 y ) 0.0028x + 0.0017 y ) 0.0031x - 0.0075 y ) 0.0063x + 0.1142

5 77 22 45 48 19 728

RAL 0 3.1/106 47.7/106 13.6/106 27.9/106 29.7/106 11.8/106 450.0/106

a The level of dG-C8-arylamine adducts were determined from a calibration line of known standards spiked into ct-DNA (500 µg) and worked up then analyzed under identical conditions to the in vitro samples (Figures 3-6). The amount of adduct has been expressed as a concentration (pmol/500 µg) and as a RAL. b Activated arylamine (0.06 mmol) was reacted with ct-DNA (10 mg). c Calibration lines were run from authentic standards (0, 20, 100, 200, 500 pg dG-C8-arylamine) added to digested ct-DNA (500 µg) and taken through the assay procedure. The correlation coefficient, R2, calculated from the linear regression line was greater than 0.995.

Table 3. Total Yield of dNs (µmol/Digest) Determined in Different Amounts of Digested ct-DNA dN pairs (%)

amount of ct-DNA (µg)

dNs in ct-DNA digest (µmol)

yield (%)

GCb

ATc

250 500 1000

756 ( 36.9 1451 ( 45.9 2649 ( 86.1

98.5 94.3 86.1

41.4 41.3 40.6

58.6 58.7 59.4

a The data are an averaged value ( SD, determined from three independent digestions. Theoretical yields were calculated assuming that the commercial ct-DNA was 95% pure. The mole percent of dN pairs GC and AT in ct-DNA was determined. b Theoretical mole percent of GC in ct-DNA ) 41.9. c Theoretical mole percent of AT in ct-DNA ) 58.1.

increased only slightly, ranging from 3.7 to 13.1%. This confirmed that the matrix had very little effect on the precision of the assay. The integrity of the dNs, released in the ct-DNA digest, was confirmed by the absence of peaks in the UV chromatogram at retention times of 2.7, 3.7, 5.1, or 6.1 min, corresponding to bases C, G, T, and A. There were no peaks observed in the UV chromatogram, corresponding to 5′P-dN (5′P-dC ) 1.6, 5′P-dG ) 2.5, 5P′-dA ) 3.3 min) or ribonucleosides (rC ) 3.2, rG ) 3.8, rU ) 7.3, and rA ) 16.3 min). DNA Concentration Limit for the Adduct Determination. ct-DNA was dissolved in H2O at a concentration of 100 µg/10 µL. Aliquots of this solution were spiked with 4CA in vitro modified DNA (32 µg). An appropriate volume of ct-DNA was taken and adjusted to 200 µL with H2O, to give final concentrations of 250, 500, and 1000 µg DNA per 200 µL. The samples were spiked with dG-C8-4ABP as internal standard (508 pg) and then sequentially digested to dNs with DNase I, NP1, and AP, in a final volume of 615 µL. An aliquot of DNA (20 µL) was analyzed by HPLC (see above). The yields obtained for the dNs are summarized in Table 3. The combined yield of dNs determined in 250 µg of digested ct-DNA fell within the error associated with the assay. For 500 µg of digested ct-DNA, the combined yield of dNs deviated by 5.7% from the theoretical value and for 1000 µg, it deviated by 13.9%. The best yields are obtained with 250 and 500 µg of DNA. For the determination of the released dG-C8-4CA, the bulk of the remaining DNA digest (595 µL) was extracted three times with EtOAc and reconstituted with 400 µL of H2O containing recovery standard dG-C8-26DMA (100 pg). The samples were analyzed by HPLC-ESI-MS/MS. The response factor for dG-C84CA was calculated from the relative peak area of dG-C8-4CA with respect to the recovery standard dG-C8-26DMA. The yield of dG-C8-4CA decreased with increasing amounts of ct-DNA. The response factor for dG-C8-4CA from 250, 500, and 1000 µg of ct-DNA was 0.71, 0.68, and 0.58, respectively. The response factor for dG-C8-4CA from 500 and 1000 µg of ct-DNA, calculated as a percent of the value from 250 µg of ct-DNA, was 96 and 82%, respectively. These percents correlated well with the combined yield of dNs determined in 500 and 1000 µg of digested

ct-DNA (Table 3). The ratio between the integrated peak areas of dG-C8-4CA and dA4CA remained constant (2.1) at each concentration of ct-DNA analyzed. Separation of DNA Adducts from Constituents of the ct-DNA Digest. Experiments were carried out to qualitatively assess the level of nonmodified dNs carried over during the adduct enrichment step. A standard solution of dG-C8-4CA (10 ng) was spiked into 250 or 500 µg of ct-DNA that had been digested to corresponding dNs. The samples were extracted with EtOAc or H2O-saturated butanol (1 × 500 µL, 2 × 250 µL). The samples were evaporated down to 5 µL and then reconstituted in MeOH (20 µL) for subsequent analysis by HPLC coupled with DAD detection (HP Hypersil BDS RP-C-18, 125 mm × 2 mm, 3 µm, linear gradient from 10% MeOH to 90% MeOH in AF over 20 min, λ ) 265 nm). For the EtOAc extracts, baseline resolution of dG-C8-4CA (14.8 min) was obtained from the nonmodified dNs (1.7-5.6 min), carried over from either 250 or 500 µg of extracted ct-DNA. The bulk of nonmodified dNs had eluted by 5.5 min, and trace levels were not detected in the UV chromatogram after 10 min. For the butanol extracts, the dG-C84CA standard eluted separately (15.2 min) from the bulk of the nonmodified waste (1.6-5.2 min). However, the baseline was not down to zero at 15 min. There appears to be low levels of nonmodified dNs bleeding from the column. It was not possible to prevent low levels of the nonmodified dNs eluting over the time range 10-20 min with the HPLC column described. Alteration of the chromatography resulted in a marked increase in retention time of dG-C8-4CA and considerable peak broadening. When a wider bore column was used, the level of nonmodified dNs detected in the latter part of the UV chromatogram decreased but the sensitivity was lost with respect to detection of the dG-C8-4CA adduct. The level of nonmodified dNs carried over during extraction increased as the amount of digested ctDNA was increased. Adduct Recoveries from EtOAc and Butanol Enrichment. Experiments were performed to determine the percentage of analyte loss during the evaporation phase for adducts spiked into either H2O-saturated butanol or EtOAc. Standard solutions of dG-C8-2MA (100 pg) or dG-C8-4CA (100 pg) were spiked directly with internal standard dG-C8-4ABP (508 pg) into 1 mL of solvent (EtOAc- or H2O-saturated butanol). The samples were evaporated as they stood or were spiked with 10 µL of DMSO or 100 µL of H2O prior to evaporation. The solvent samples without H2O or DMSO were evaporated to 20 µL. The DMSO spikes were evaporated to 10 µL, and the water spikes were evaporated in vacuo to 80 µL. The samples were reconstituted with H2O containing recovery standard dG-C8-26DMA (100 pg) and then injected onto the HPLC-ESI-MS/MS system. The results have been presented in Table 4. The overall recovery of dG-C8-2MA and dG-C8-4CA, at concentrations of 20 and 100 pg from digested ct-DNA (500 µg), was then investigated. Standard solutions of dG-C8-2MA (20 or 100 pg) or dG-C8-4CA (20 or 100 pg) were spiked into digested ct-DNA (500 µg) with internal standard dG-C8-4ABP (508 pg).


Jones and Sabbioni flow rate of 0.25 mL/min). The determination limits for each of the dG-C8-arylamine standards, with a signal-to-noise ratio greater than 40, were as follows: 50, 50, 26, 26, 52, 52, and 46 fmol/500 µg DNA for 2CA-, 4CA-, 2MA-, 4MA-, 24DMA-, 26DMA-, and 4ABP-dG, respectively. If arylamine adducts were not detected in rat DNA hydrolysates extracted with EtOAc, H2O-saturated butanol was used.

Table 4. Recoveries of dG-C8-2MA Standard (100 pg), Following Evaporation of the Solvents, Were Determineda conditions

dG-C8-2MA/dG-C8-26DMA

standards injected directly EtOAc only EtOAc + H2Ob EtOAc + DMSOc butanol butanol + DMSOc butanol + H2Ob

2.96 ( 0.083 0.54 ( 0.074 2.31 ( 0.189 1.49 ( 0.120 1.04 ( 0.031 1.40 ( 0.177 1.86 ( 0.209

Results and Discussion

a

The response ratio of dG-C8-2MA with respect to dG-C826DMA was compared to the same standards injected directly onto the HPLC-ESI-MS/MS. b These samples were reduced to approximately 80 µL prior to reconstitution. c The samples were reduced to 10 µL prior to reconstitution.

The samples were extracted by vortex mixing for 1 min with either H2O-saturated butanol or EtOAc (1 × 500 µL, 2 × 250 µL). The samples were then spiked with H2O (100 µL) and reduced in vacuo to approximately 80 µL. The samples were then reconstituted in H2O (100 µL) containing recovery standard dG-C8-26DMA (100 pg). The results are presented in Table 5. The best recoveries were obtained with butanol. The precision associated with determination of the dG-C8-arylamine adducts at the lowest calibration point (20 pg) and five times this value (100 pg) was 18.7 and 10.9% RSD, respectively. Experiments were performed to evaluate the percent of residual butanol that could be tolerated by the analytical system. A standard solution (25 µL) containing dG-C8-2MA (20 pg), dG-C8-26DMA (100 pg), and dG-C8-4ABP (508 pg) was spiked into aliquots of H2O (370 µL), and butanol was added to give relative percentages of 0.5, 1, 2, and 4%. When the samples were injected onto the chromatographic system, substantial peak splitting occurred when butanol was present at either 4 or 2%. The splitting was less obvious at 1%, and there was no observable splitting when present at less than 1%. Therefore, all butanol extractions were reconstituted in a larger volume of H2O (total volume of 500 µL) prior to analysis by HPLC-ESIMS/MS. Purification and Enrichment of Rat Liver DNA Adducts by Liquid-Liquid Partitioning. DNA hydrolysates (600 µL), in 2 mL Teflon tubes, were extracted three times with H2O-saturated butanol (1 × 500 µL and 2 × 250 µL), by vortex mixing for 2 min each time. After each extraction, the samples were centrifuged for 10 min at 2000g. The organic phases were combined, spiked with H2O (100 µL), and then reduced at room temperature in a speed evaporator, to approximately 80 µL. The samples were transferred to glass vials (32 mm × 12 mm) and made up to a total volume of 500 µL. The samples were manually injected onto an HPLC system, consisting of an HPLC pump (Hewlett-Packard series 1050), a mobile phase degasser (degasys DG-1310), and a quadrupole ion trap system (Thermo Finnigan LCQ-Duo) used in the ESI mode. Positive ions were detected for each compound of interest, and the m/z of the parent [M + H]+ and daughter ion [BH2]+ were studied. The compounds were separated on a Hypersil BDS C-18 column (125 mm × 2 mm, 3 µm) with the following conditions: 5 min isocratic elution with 10% MeOH in 10 mM AF buffer and then a linear 20 min gradient; 10% MeOH in 10 mM AF buffer to 90% MeOH with a

Synthesis of DNA Adducts. DNA adducts of arylamines were synthesized by reaction of each N-acetoxyarylamine with dG and dA. The N-acetoxyarylamines were generated, but not isolated, by reaction of the corresponding N-hydroxyarylamines with acetyl cyanide (AcCN) (Figure 1). A solution of each N-acetoxyarylamine (2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, and 4ABP) was added to an aqueous solution of dG or dA. The resulting mixture was initially purified by solvent extraction, to remove the bulk of byproducts generated in each reaction. The synthesized adducts were further purified and enriched by preparative TLC. Each discrete band of UV active silica (254 nm) was removed and extracted with MeOH. An aliquot of each extracted silica zone was analyzed in MS/MS mode following positive ESI. The extracted silica zones that gave correct MS/MS spectra for each adduct were kept (Table 1). The m/z of these ions were consistent with formation of the protonated molecular ion [M + H]+ and daughter ion, [BH2]+. The [BH2]+ fragment corresponded to the loss of the deoxyribose sugar [M + H - 116]+, resulting from cleavage of the glycosidic bond. In most instances, additional fragments of [M + H 17]+ and [M + H - 18]+ were observed in the MS/MS spectra of the dA-arylamine adducts with a relative abundance smaller than 20%. These fragments were also seen in some of the dG-arylamine adducts. These fragments have tentatively been assigned as loss of the A [G] NH2 group and the A [G] N1 hydrogen, for ions [M + H - 17]+, and loss of H2O, for ions [M + H - 18]+. However, it appears that the [M + H - 18]+ fragment is both concentration- and matrix-dependent. At higher concentrations, the [M + H - 18]+ peak is not apparent. Selected ion chromatograms for each arylamine adduct have been presented as follows: 2CA and 4CA (Figure 2); 24DMA and 26DMA (Figure 3); 2ABP and 3ABP (Figure 4); 2MA and 4MA (Figure 5); and 4ABP (Figure 6). Following adduct characterization by ESI-MS/MS, the CE was optimized to achieve maximum formation of the [BH2]+ daughter ion from CID of the [M + H]+ molecular ion. This was carried out to enhance the performance of the mass analyzer in terms of MS/MS sensitivity. In most instances, application of 40% CE for the dG-arylamine adducts and 40% for the dA-arylamine adducts gave

Table 5. Recoveries of dG-C8-Arylamine Standards Spiked into Digested ct-DNA (500 µg) and Worked up by Extraction with Either EtOAc- or H2O-Saturated Butanola conditions standards solutions injected directly EtOAc only EtOAc with NaCl butanol

dG-C8-2MA (20 pg)

dG-C8-2MA (100 pg)

dG-C8-4CA (20 pg)

dG-C8-4CA (100 pg)

0.319 ( 0.058

2.196 ( 0.02

0.055 ( 0.0049

0.234 ( 0.013

0.039 ( 0.008 0.083 ( 0.003 0.114 ( 0.017

0.314 ( 0.059 0.612 ( 0.074 0.904 ( 0.109

0.004 ( 0.0005 0.014 ( 0.002 0.019 ( 0.0021

0.028 ( 0.0031 0.070 ( 0.0076 0.089 ( 0.0082

a The response ratio of dG-C8-2MA or dG-C8-4CA with respect to dG-C8-26DMA (standard added after work up) was compared to nonextracted standards injected directly onto the HPLC-ESI-MS/MS.



Figure 1. Synthesis of arylamine-DNA adducts by reaction with N-acetoxyarylamines. Panel 1: Generation of the active electrophile of each arylamine through reaction of the corresponding N-hydroxyarylamine with AcCN. Panel 2: Reaction of the N-acetoxyarylamine with dG. The products correspond to the dG-C8-arylamine adduct, except for dG2ABP and dG3ABP, whose structures have not been elucidated. Panel 3: Reaction of the N-acetoxyarylamine with dA. The position of covalent interaction between the pyrimidine base and the N-acetoxyarylamine was not determined and has been presented as a bracketed structure.

optimum formation of the [BH2]+ ion, except for dG-2MA, -4MA, -24DMA, and -26DMA for which 30% CE was optimal. The purity of each silica zone, which gave the correct MS/MS spectra, was analyzed by HPLC-ESI-MS/MS. One major peak was observed for dG2ABP, dG3ABP, and dG4ABP. This confirmed that the N-acetoxyarylamines of 2ABP, 3ABP, and 4ABP had covalently bonded to one position within the dG molecule, under the described reaction conditions. The UV absorbance spectra of the purified dG4ABP adduct, in MeOH, was consistent with that reported in refs 38 and 39 and was assigned as the dG-C8-4ABP. The concentration of dG-C8-4ABP was calculated from its molar extinction coefficient at 305 nm (31 000) (39). The other dG-C8-arylamine adducts had been characterized in ref 34. Typically, more than one peak was observed, with correct MS/MS spectra, in the full scan chromatogram of each dA-arylamine adduct. This indicated that the N-acetoxyarylamines had covalently bonded to more than one position within the dA molecule, under the described reaction conditions. The retention times for the synthesized dG and dA-arylamine adducts have been reported in Table 1, alongside the adducts determined from the analysis of digested ct-DNA, which had been modified in vitro with each N-acetoxyarylamine. Each adduct (dG2ABP, dG3ABP, dG-C8-4ABP, dA2CA, dA4CA,dA2MA, dA4MA, dA24DMA, dA26DMA, dA2ABP, dA3ABP, and dA4ABP) had been synthesized on a micromolar scale. Quantitative yields of each adduct were not obtained, and structural characterization of the covalent interaction between the arylamine and the dG or dA were not achieved by NMR. In Vitro Reaction of ct-DNA with N-Acetoxyarylamines. Experiments were performed to determine whether the N-acetoxyarylamines covalently bonded to the same positions on dG and dA, within whole ct-DNA, as they did when reacted with single bases dG and dA.

Figure 2. Panel 1: HPLC/MS/MS chromatogram of digested ct-DNA modified in vitro with N-acetoxy-2CA. The adducts dC2CA (1a), dG-C8-2CA (1b), dT2CA (1c), and dA2CA (1d) and the internal standard dG-C8-4ABP (1e) were detected following ion trap CID (40% CE) of [M + H]+. Panel 2: HPLC/MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy2CA. The dG-C8-2CA (2a), the dA2CA (2b), and the internal standard dG-C8-4ABP (not shown, 19.9 min) were detected following ion trap CID (40% CE) of [M + H]+. Panel 3: HPLC/ MS/MS chromatogram of ct-DNA, which was modified in vitro with N-acetoxy-4CA and then digested with enzymes. The adducts dC4CA (3a), dG-C8-4CA (3b), dT4CA (3c), dA4CA (3d), and the internal standard dG-C8-4ABP (3e) were detected following ion trap CID (40% CE) of [M + H]+. Panel 4: HPLC/ MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy-4CA. The adducts dG-C8-4CA (4a) and dA4CA (4b) and the internal standard dG-C8-4ABP (not shown, 19.9 min) were detected following ion trap CID (40% CE) of [M + H]+.


Figure 3. Panel 1: HPLC/MS/MS chromatogram of ct-DNA, which was modified in vitro with N-acetoxy-24DMA and then digested with enzymes. The adducts dC24DMA (1a), dG-C824DMA (1b), dT24DMA (1c), dA24DMA (1d), and the internal standard dG-C8-4ABP (1e) were detected following ion trap CID (30% CE) of [M + H]+. Panel 2: HPLC/MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy-24DMA. The adducts dG-C8-24DMA (2a), dA24DMA (2b), and the internal standard dG-C8-4ABP (not shown, 20.3 min) were detected following ion trap CID (30% CE) of [M + H]+. Panel 3: HPLC/ MS/MS chromatogram of ct-DNA, which was modified in vitro with N-acetoxy-26DMA and then digested with enzymes. The adducts dC26DMA (3a), dG-C8-26DMA (3b), dT26DMA (3c), dA26DMA (3d), and the internal standard dG-C8-4ABP (3e) were detected following ion trap CID (30% CE) of [M + H]+. Panel 4: HPLC/MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy-26DMA. The adducts dG-C8-26DMA (4a) and dA26DMA (4b) and the internal standard dG-C8-4ABP (not shown, 20.3 min) were detected following ion trap CID (30% CE) of [M + H]+.

The activated N-acetoxy derivative of each arylamine (2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, and 4ABP) was reacted with ct-DNA. The DNA was purified by solvent extraction and then precipitated. Each modified DNA sample was digested to dNs by enzymatic action with NP1 and AP. The dN adducts were extracted with butanol and analyzed by HPLC-ESI-MS/MS. For each of the in vitro modification reactions between the DNA and the N-acetoxyarylamines described, peaks were observed corresponding to dC-, dG-, dT-, and dAarylamine adducts. For adducts to dG, the most prevalent peak eluted with identical retention times and mass spectra to the peak observed in the single base reaction. The retention times have been listed in Table 1 and selected ion chromatograms for 2CA, 4CA, 24DMA, 26DMA, 2ABP, and 3ABP showing adducts to dC, dG, dT, and dA have been presented in Figures 2-4. Although comparative synthetic equivalents were not available, by acquiring the [M + H]+ and [BH2]+ ions with m/z equal to the formation of an adduct between the arylamine and the dC or dT, it was possible to identify unknown adducts. Selected ion chromatograms for 4MA and 4ABP adducts are shown in Figures 5 and 6, respectively.

Jones and Sabbioni

Figure 4. Panel 1: HPLC/MS/MS chromatogram of ct-DNA, which was modified in vitro with N-acetoxy-2ABP and then digested with enzymes. The adducts dC2ABP (1a), dG2ABP (1b), dT2ABP (1c), dA2ABP (1d), and the internal standard dG-C826DMA (1e) were detected following ion trap CID (40% CE) of [M + H]+. Panel 2: HPLC/MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy-2ABP. The adducts dG2ABP (2a) and dA2ABP (2b) and the internal standard dG-C8-26DMA (not shown, 14.2 min) were detected following ion trap CID (40% CE) of [M + H]+. Panel 3: HPLC/MS/MS chromatogram of ctDNA, which was modified in vitro with N-acetoxy-3ABP and then digested with enzymes. The adducts dC3ABP (3a), dG3ABP (3b), dT3ABP (3c), dA3ABP (3d), and the internal standard dGC8-26DMA (3e) were detected following ion trap CID (40% CE) of [M + H]+. Panel 4: HPLC/MS/MS chromatogram of dG and dA modified in vitro with N-acetoxy-3ABP. The adducts dG3ABP (4a) and dA3ABP (4b) and the internal standard dG-C8-26DMA (not shown, 13.7 min) were detected following ion trap CID (40% CE) of [M + H]+.

For 4ABP, the peak observed in the single ion chromatogram of the in vitro reaction with ct-DNA and the single base reaction with dG was identified as dG-C84ABP. For 2ABP and 3ABP, the peaks observed in the in vitro reaction with ct-DNA and single base reaction with dG were not structurally characterized by 1H or 13C NMR. For the dimethylaniline compounds, 24DMA and 26DMA, we have demonstrated that the activated forms of these compounds (N-acetoxy derivatives in our study) reacted with ct-DNA in vitro to yield dA and dG adducts in ct-DNA. The predominant adduct was to the C8position of dG. The presence of dG adducts in in vitro modified DNA has been demonstrated recently by others (42, 49). When control ct-DNA was digested, extracted, and analyzed under identical conditions, no peaks were observed in the selected ion chromatograms at retention times corresponding to the dG-C8-arylamines or additional dG-arylamine adducts found in the modified ctDNA samples. The levels of dG-C8-arylamine adducts formed in ctDNA, following reaction with each N-acetoxyarylamine, were quantified against calibration lines of authentic dGC8-arylamines standards and have been listed in Table 2. The lowest modification level was detected in ct-DNA that had been modified with N-acetoxy-2CA. The highest


Figure 5. Panel 1: HPLC/MS/MS chromatogram of digested hepatic DNA (500 µg) from a rat dosed with 0.5 mmol/kg 4MA. The internal standard dG-C8-2MA (1a), the recovery standard dG-C8-4CA (not shown, 17.8 min), and the adducts dG4MA (1a) and dA4MA (1b) were detected. Panel 2: HPLC/MS/MS chromatogram of digested hepatic DNA (500 µg) from a rat dosed with 0.5 mmol/kg 4MA-d4. The internal standards dG-C8-2MA/ dG-C8-4MA (2c), the recovery standard dG-C8-4CA (not shown, 17.8 min), and the adducts dG4MA-d4 (2a) and dA4MA-d4 (2b) were detected. Panel 3: HPLC/MS/MS chromatogram of digested hepatic DNA (500 µg) from a control rat dosed with vehicle only. The masses corresponding to the following adducts were monitored with CID (30% CE): dG4MA-d4 (3a), dA4MAd4 (3b), dG4MA (3c), dA4MA (3d), and the internal standard dG-C8-4CA (3e). Panel 4: HPLC/MS/MS chromatogram of ctDNA, which was modified in vitro with N-acetoxy-4MA and then digested with enzymes.

level of modification was detected in ct-DNA that had been modified with N-acetoxy-4ABP. The levels of dG adducts in ct-DNA reacted with the para-substituted arylamines were higher than the levels detected resulting from reaction with the ortho-substituted arylamines. The adduct levels in the 24DMA-modified ct-DNA were more than 2-fold higher than those found with the 26DMAmodified ct-DNA. The level of adducts for each of the above arylamines, except 4ABP, fell within a factor of 100. This inferred that arylamines bearing a methyl group at the para position to the arylamine nitrogen were better able to stabilize the putative nitrenium ion intermediate through inductive effects, as compared to ortho methyl substituents (43). Our results were in good agreement with those of Marques et al. who compared the yields of the activated arylamines with dG (44). The adduct levels determined for dG-C8-4CA could not be explained in terms of electronic stabilization effects provided by the halogen substituent group for the nitrenium ion intermediate. Chloro groups have been shown to be electron withdrawing through inductive effects and only weakly electron donating through resonance stabilization effects and thus would not be predicted to stabilize the positive charge on the nitrenium ion as well


Figure 6. Panel 1: HPLC/MS/MS chromatogram of ct-DNA, which was modified in vitro with N-acetoxy-4ABP and then digested with enzymes. The adducts dG-C8-4ABP (1a), dA4ABP (1b), and the internal standard dG-C8-4CA (not shown, 17.2 min) were detected following ion trap CID with 40% CE. Panel 2: HPLC/MS/MS chromatogram of digested hepatic DNA (130 µg) from a rat dosed with 0.5 mmol/kg 4ABP. The adducts dGC8-4ABP (2a) and dA4ABP (2b), the internal standard dG-C84CA (2c), and the recovery standard dG-C8-26DMA (2d) were detected following ion trap CID with 40% CE. Panel 3: HPLC/ MS/MS chromatogram of digested hepatic DNA (150 µg) from a rat dosed with 0.5 mmol 4ABP-d9. The adducts dG-C8-4ABPd9 (3a) and dA4ABP-d9 (3b), the internal standard dG-C8-4CA (3c), and the recovery standard dG-C8-26DMA (3d) were detected following ion trap CID with 40% CE. Panel 4: HPLC/ MS/MS chromatogram of digested hepatic DNA (500 µg) from a rat dosed with 0.5 mmol/kg 4NBP. The adducts dG-C8-4ABP (4a) and dA4ABP (4b), the internal standard dG-C8-4CA (4c), and the recovery standard dG-C8-26DMA (not shown, 14.6 min) were detected following ion trap CID with 40% CE.

as arylamines with methyl substituents. The relatively low RAL value determined for 2CA (RAL ) 3.1) agreed with this assumption. For 4CA, the RAL value (RAL ) 48) was much higher than expected and may have been due to the lipophilicity of the compound, which might facilitate its intercalation into the double helix of the ctDNA. The same lipophilicity is present in 2CA; however, ortho substitution is a steric hindrance for the nucleophilic attack on the nitrenium ion. In conclusion, identification of these adducts in vitro provided valuable information about the relative reactivity of each arylamine to nucleophilic sites in DNA. The selected ion chromatograms of the in vitro modification reactions revealed that the arylamines had reacted and covalently bonded to more than one nucleoside. In addition to the formation of dG adducts, covalent binding to positions on dC, dT, and dA were identified by HPLC-ESI-MS/MS. For each compound of interest, multiple peaks were observed in the selected ion chromatograms of the digested ct-DNA. With respect to dAarylamine adducts, these peaks had identical retention times and MS/MS spectra to the peaks observed in the selected ion chromatograms of single base reactions


between dA and the N-acetoxyarylamines. The retention times of these peaks have been listed in Table 1. The elution of these adducts followed the same pattern as described for the dG-arylamine adducts. Optimization of DNA Adduct Determination. To obtain the best yield in the in vivo samples, parameters were tested as follows: (i) optimization of enzyme combination (2-4 enzymes), (ii) DNA concentration limit (100-1000 µg), and (iii) adduct recoveries from liquidliquid extraction (EtOAc or butanol). The results are presented in detail in the Experimental Section and in Tables 3-5. In summary, the highest adduct yields are obtained using 250-500 µg of DNA, four enzymes for the DNA digestion, and butanol for adduct enrichment. The disadvantage of using butanol instead of EtOAc is the carry over of unmodified nucleosides, which might interfere with the ESI-MS/MS detection of the adducts. In addition, it could be shown that butanol residues larger than 2% cause peak splitting. Determination of DNA Adducts in Livers of Exposed Rats (Table 6). Female rats (two per group) were dosed orally with a range of structurally related arylamines and nitroarenes (0.5 mmol/kg) and sacrificed after 24 h. Isolated liver DNA was spiked with internal standard (100 pg) and then sequentially digested to individual dNs with DNase I and NP1 then SVPD with AP, extracted with H2O-saturated butanol, and analyzed by HPLC/MS/MS. In rats dosed with 4ABP, 4ABP-d9, and 4NBP, two major peaks were present in the chromatograms of digested hepatic DNA. These peaks corresponded to an adduct with dG and an adduct with dA (Figure 6, panels 2a,b,3a,b,4a,b). The dG4ABP peak had an identical retention time (20.1 min) to the peak found in digested N-acetoxy-4ABP in vitro modified ct-DNA (Figure 6, panel 1a) and to the analogous dG-C8-4ABP authentic standard. The dA4ABP peak had an identical retention time (21.5 min) to the later eluting peak found in digested N-acetoxy-4ABP in vitro modified ct-DNA. This dA4ABP peak found in digested rat DNA and modified ct-DNA (21.5 min) had a different retention time to the peak observed in the selected ion chromatogram of the single base reaction between dA and N-acetoxy4ABP (19.6 min). There were no peaks corresponding to a dG-C8-4ABP, dG-C8-4ABP-d9, or dA4ABP adduct observed in selected ion chromatograms of digested hepatic DNA from control rats dosed with vehicle only (1,2-propanediol). The level of dG-C8-4ABP was determined by comparison to a calibration line of dG-C8-4ABP. Standard solutions of dG-C8-4ABP (0, 23, 102, 204, 508, and 1016 pg) were spiked into digested ct-DNA with internal standard dG-C8-4CA (100 pg) and then worked up and analyzed under identical conditions to the rat DNA samples. The level of adducts (fmol/500 µg hepatic DNA) was 8010 ( 1100, 4900 ( 710, and 120 ( 15 in rats dosed with 4ABP, 4ABP-d9, and 4NBP, respectively. The discrepancy in adduct levels from rats dosed with the deuterated and nondeuterated forms of 4ABP was due to the fact that the deuterated adduct was quantitated from a calibration line of dG-C8-4ABP in the absence of a synthetic deuterated standard. Each value was an average ( the SD determined in hepatic DNA from two dosed rats analyzed three times. The level of adducts was higher (66-fold) in rats dosed with 4ABP as compared to 4NBP. The experiment with 4ABP-d9 confirmed the formation of the adducts seen with 4ABP and 4NBP. The adduct levels (fmol/500 µg hepatic DNA) were

Jones and Sabbioni Table 6. Arylamine Adducts Covalently Bonded to Hb and to dG in Digested Hepatic DNA of Rats Administered a Single Bolus of Arylamine or Nitroarene (0.5 mmol/kg) arylamine administered to rats

dG-arylamine

1,2-propanediol 2CNB 2CA 4CNB 4CA 2NT 2MA 2MA-d4 4NT 4MA 4MA-d4 24DMA 24DMA-d3 26DMA 26DMA-d3 2ABP 3ABP 4ABP 4ABP-d9 4NBP

nda nd nd nd nd possibleb,c possibleb,c possibleb,c possibleb,c possibleb,c possibleb,c nd nd nd nd nd nd dG-C8-4ABP dG-C8-4ABP dG-C8-4ABP

hepatic DNA RAL (× 10-8)

MS Following In Vitro

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