Hemoglobin and DNA adducts in rats exposed to 2-nitrotoluene
Christopher R. Jones1,2,
Armin Beyerbach1,
Wolfgang Seffner3 and
Gabriele Sabbioni1,4
1 Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, Nussbaumstrasse 26, D-80336 München, Germany
2 Department of Environmental and Occupational Medicine, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
3 Institut für Wasser-, Boden- und Lufthygiene, Forschungsstelle Bad Elster, Bad Elster, Germany
4 To whom correspondence should be addressed at: Institute of Environmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland Email: gabriele.sabbioni{at}bluewin.ch
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Abstract
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2-Nitrotoluene (2NT) is an important commercial chemical intermediate. A recent National Toxicology Programme (NTP)-study demonstrated clear evidence of carcinogenic activity of 2NT in rats. In the present study male WELS-Fohm rats were dosed chronically with 2NT, 5 days a week for 12 weeks. Hemoglobin (Hb) adducts and hepatic DNA adducts were analyzed. After mild base treatment of Hb, 2-methylaniline (2MA) was released and quantified using gas chromatography/mass spectrometry. 2'-Deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts of 2MA were found in hepatic DNA using electrospray-mass spectrometry (ESI-MS/MS). The dG adduct found in vivo did not co-elute with N-(2'-deoxyguanosine8-yl)-2-methylaniline which is the expected adduct for arylamines. The dG adduct detected in the dosed rats was not present in calf thymus-DNA (ct-DNA) modified in vitro with N-acetoxy-2MA. The dA adduct detected in rats was a very minor product in ct-DNA modified in vitro. The dG and dA adducts found in the 2NT-dosed rats increased with the dose. The same increase was seen for the Hb adduct levels measured in the same animals. The increase of DNA and Hb adduct levels were supralinear. There was a very strong linear relationship between the level of dG-2MA adducts and dA-2MA adducts in hepatic DNA from rats administered 2NT over the whole dose range studied (r2 = 0.9). A strong linear relationship also existed between the level of dG-2MA or dA-2MA adducts, in hepatic DNA, and Hb adducts, over the whole dose range (r2
0.9). Thus, there was strong evidence to support the notion that Hb adducts were an effective surrogate marker for the hepatic DNA damage of rats chronically administered 2NT.
Abbreviations: A, adenine; AF, ammonium formate; AP, alkaline phosphatase; dA, 2'-deoxyadenosine; C, cytosine; CE, collisional energy; CID, chemical induced dissociation; ct-DNA, calf thymus deoxyribonucleic acid; ESI-MS/MS, electrospray-mass spectrometry; dC, 2'-deoxycytidine monohydrochloride; dG, 2'-deoxyguanosine; DNase I, deoxyribonuclease I; 5'P-dG, 2'-deoxyguanosine-5'-monophosphate; dG-C8-2MA, N-(2'-deoxyguanosine8-yl)-2-methylaniline; dG-C8-4ABP, N-(2'-deoxyguanosine8-yl)-4-aminobiphenyl,; dN, 2'-deoxyribonucleosides; dG-C8-4CA, N-(2'-deoxyguanosine8-yl)-4-chloroaniline; dT, thymidine; EI, electron-impact ionization; G, guanine; Hb, hemoglobin; 2MA, 2-methylaniline; NP1, nuclease P1; 2NT, 2-nitrotoluene; NTP, National Toxicology Programme; PFPA, pentafluoropropionic anhydride; rA, adenosine; rC, cytidine; rG, guanosine; rU, uridine; SIM, single-ion monitoring; T, thymine; 245TMA, 2,4,5-trimethylaniline; tR, retention time
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Introduction
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2-Nitrotoluene (2NT) is an important commercial chemical used to synthesize agricultural chemicals, rubber chemicals and azo and sulfur dyes (1). An estimated 2028 million kilograms of 2NT were produced in the US in 1990 (2). Environmental surveys have demonstrated the presence of 2NT in rivers and in drinking water (3,4). A recent NTP study demonstrated clear evidence of carcinogenic activity of 2NT in both male and female F344 rats. In male rats increased incidences of malignant mesothelioma, subcutaneous skin neoplasms, mammary gland fibroadenoma and liver neoplasms were observed (5).
Data on human exposure to 2NT in the workplace were not available. However, the Occupational Safety and Health Administration have set an 8-h, time weighted average (TWA) permissible exposure limit of 5 p.p.m. (30 mg/m3) for nitrotoluenes (6) and the American Conference of Governmental Industrial Hygienists recommended a threshold limit value of 2 p.p.m. (11 mg/m3) for the 8-h TWA. Although clinical data have cited methemoglobin formation as a symptom of 2NT exposure, there is no human biological exposure data available on 2NT.
The metabolism of 2NT administered orally was studied in F344 rats (5). The rate of excretion of 2NT in rats was such that 90% of the administered radioactivity was excreted in urine within 72 h. The majority of 2NT metabolites detected in excreted rat urine were the result of oxidation of the methyl group to the nitrobenzyl alcohol, which underwent further oxidative biotransformation. The major metabolites excreted in the urine of male F/344N rats after a single oral dose of 200 mg/kg 2NT were 2-nitrobenzoic acid (21.0% of the dose), 2-nitrobenzyl glucuronide (16.6%), 2-aminobenzyl alcohol (18.2%), S-(2-nitrobenzyl)-N-acetylcysteine (2-nitrobenzylmercapturic acid) (10.4%), 2-nitrobenzyl alcohol (1.8%) and 2-methylaniline (2MA) (1.3%). Female rats produced a similar profile of major metabolites, with the exceptions of 2-aminobenzyl alcohol and S-(2-nitrobenzyl)-N-acetylcysteine which were excreted at significantly lower levels than in males. Comparable results were obtained by the group of Rickert (710).
For the present study we investigated the hemoglobin (Hb) and DNA adducts present in chronically dosed rats (Figure 1). Nitroarenes are reduced mainly in the intestine and partially in the liver (10) to arylamines. Therefore, the same Hb adducts are formed as after dosing animals with arylamines. Usually the levels of Hb adducts are larger in rats given arylamines (15). Arylamines are oxidized to N-hydroxyarylamines which are further oxidized to nitrosoarenes in the erythrocytes and then form sulfinamide adducts with cysteine residues in Hb. N-Hydroxyarylamines can be further activated to highly reactive N-O-ester intermediates such as O-acetylates or sulfates which are capable of reacting with DNA (1113). Hb adducts have been found after a single dose of 2NT or 2MA (14,15). Hb adduct levels were
10 times larger in animals administered 2MA (= 0.063% of the total dose bound to Hb). In addition, adducts resulting from the oxidation of the methyl group in 2NT are possible (Figure 1). To date, no DNA adducts have been found and characterized in animals administered 2MA or 2NT.
In general, the predominant arylamine DNA adduct found in vivo is a reaction product at the C8 position of 2'-deoxyguanosine (11,13). Therefore, if sulfinamide adducts with Hb are present one might postulate that the intermediate, N-hydroxy-2-methylaniline, might interact with DNA to yield, according to present knowledge, N-2'-(deoxyguanosin-8-yl)-2-methylaniline (dG-C8-2MA). The goal of the present work is the investigation of DNA and Hb adducts in rats given 2NT chronically.
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Materials and methods
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Chemicals
Calf thymus deoxyribonucleic acid sodium salt (ct-DNA) (95%), 2'-deoxycytidine monohydrochloride (dC), thymidine (dT), 2'-deoxyguanosine-5'-monophosphate (5'P-dG) and ethanol (spectrophotometric grade) were obtained from Aldrich (Taufkirchen, Germany). Nuclease P1 (NP1) from 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). 2'-Deoxyguanosine (dG) monohydrate, formic acid (puriss. p.a.), magnesium chloride hexahydrate (Microselect, MgSO4), mannitol (Microselect grade) zinc sulphate (puriss. p.a., ZnSO4), zinc chloride (Microselect grade, ZnCl2), sodium acetate (NaOAc), tri-sodium citrate dihydrate (puriss. p.a., sodium citrate), triethylamine (puriss. p.a., NEt3), pyruvonitrile, ammonium formate (Microselect grade, AF), succinic acid disodium salt anhydrous purum (succinic acid) and tris(hydroxymethyl) aminomethane (Microselect grade, Tris) were obtained from Fluka (Deisenhofen, Germany). Dithiothreitol was obtained from Diagnostic Limited (CT). 2-Propanol (SupraSolv grade), Tritriplex EDTA p.a., sodium dodecyl sulphate salt (>99%, SDS), 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid >99% (HEPES), NaCl p.a., sucrose, chloroform (SupraSolv grade, CHCl3) and LiChrosolv HPLC grade water were obtained from Merck (Darmstadt, Germany). Glycerol p.a. and phenol [redistilled phenol, equilibrated in and covered with 10 mM Tris, 1 mM EDTANa2 (pH 7.58.0)] were obtained from Roth (Karlsruhe, Germany). Cytidine (rC), adenosine (rA), cytidine (rC), guanosine (rG), uridine (rU), cytosine (C), adenine (A), thymine (T) and guanine (G) were obtained from Serva (Heidelberg, Germany). 2'-Deoxyadenosine (dA), deoxyribonuclease I (DNase I) type II from bovine pancreas, alkaline phosphatase (AP) type III from Escherichia coli (cat. no. P4252), 2'-deoxycytidine-5'-monophospate (5'P-dC), 2'-deoxyadenosine-5'-monophosphate (5'P-dA) and bis [2-hydroxyethyl] iminotris [hydroxymethyl] methane (BisTris) were obtained from Sigma (Taufkirchen, Germany). Acetonitrile (Baker-ultra-gradient HPLC grade), methanol (Baker ultra resi analyzed) and water (Baker ultra resi-analyzed) were obtained from Baker (Griesheim, Germany). N-(2'-deoxyguanosine8-yl)-2-methylaniline (dG-C8-2MA), N-(2'-deoxyguanosine8-yl)-4-chloroaniline (dG-C8-4CA), N-(2'-deoxyguanosine8-yl)-4-aminobiphenyl (dG-C8-4ABP) have been synthesized according to Beyerbach et al. (18). N-(2'-deoxyguanosine8-yl)-4-aminobiphenyl (dG-C8-4ABP) has been synthesized according to Famulok et al. (19).
Mass spectrometry
Helium and nitrogen of 99.999% purity were used (Linde, Munich, Germany). Gas-chromatography/mass spectrometry (GC/MS) analyses were carried out on a Hewlett Packard GC (HP 5890II) interfaced with a mass spectrometer (HP 5989A). The HPLC/MS/MS analyses were performed on an ion trap mass spectrometer (LCQ-Duo, Thermo Finnigan, San Jose, CA) employing positive electrospray chemical ionization (ESI). The following instrument parameters were applied: capillary temperature, 220°C, nitrogen sheath gas flow, 80 (arbitrary units); and auxiliary gas flow 0 (arbitary units); spray voltage, 5 kV. The remaining parameters were optimized by the autotune program to achieve maximum transmission of the [M ± H]± ion of dG-C8-2MA, using 100 pg/ml of dG-C8-2MA, 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 collision-induced decay. A collision energy was selected that optimized fragmentation of the [M ± H]± parent ion to the daughter ion [BH2]±.
Animal experiment
The experiments were performed with male rats (1.0 F1 BD IXx0.1 WELS Fohm) in Bad Elstar, Germany. At the beginning of the study the rats were 4 months old. Twelve rats per group and six rats per cage were set up for the experiment. The animals were given feed (Sniff) ad libitum. 2NT (15 g) was dissolved in sunflower oil (50 ml) and aliquots thereof (0.6, 1.7 and 5 ml) were added to 250 ml 1% Tween-80 solution in a water bottle. The suspension was shaken four times per day. The animals only had access to the water bottle during the day. The consumed water was measured daily. This resulted in a daily dose of 40 (0.29), 96 (0.70) and 250 mg (1.82 mmol)/kg. The experiment was performed for 12 weeks.
Isolation of DNA from rat liver
DNA was isolated and purified from rat liver essentially by the procedure described in the literature (16,17). Liver samples (3 g) were thawed at room temperature and homogenized in 15 ml buffer, pH 7.4 (250 mM mannitol, 70 mM sucrose and 5 mM HEPES) in a 30 ml potter. The homogenized tissue was transferred to a teflon tube (30 ml) and centrifuged at 1000 g for 10 min. The supernatant was removed to waste and the pellet re-suspended in 15 ml of buffer (1% SDS, 1 mM EDTA). Protein was removed by addition of Proteinase K (7.5 mg) with incubation overnight at 37°C. TrisHCl, pH 7.4 (1.5 ml), was added to the aqueous phase which was sequentially extracted with phenol (15 ml), then equal volumes of 25:24:1 phenol:CHCl3:2-propanol and 24:1 CHCl3:2-propanol. DNA was precipitated from the aqueous phase with 0.1 vol of 5.0 M NaCl and 2 vol of EtOH (-20°C) overnight at -20°C. The DNA pellet was collected by centrifugation at 1000 g for 10 min then washed with 70% EtOH (1 ml). The DNA was re-dissolved in 6 ml buffer (1.5 mM NaCl, 0.15 M sodium citrate, 1 mM EDTA), then 1.0 M TrisHCl pH 7.4 (300 µl), RNase A (600 µg) and RNase T1 (300 U) were added, and the mixture incubated for 30 min at 37°C. The mixture was extracted once with an equal volume of 24:1 CHCl3:2-propanol. The DNA was re-precipitated from the aqueous phase as before. The DNA was redissolved in water at
2 mg/ml and stored at -20°C.
Determination of DNA concentration
The concentration and purity of each extracted DNA sample was determined by UV spectrometry. Dilutions (1/50) of thawed DNA solutions were made in H2O and pipetted into quartz cuvettes. Their absorbance was read between 220 and 300 nm wavelength. The maximum UV absorption was found at 260 nm. The DNA concentration was calculated at absorbance A260/280 assuming 50 µg/ml = 1.0 absorbance unit at 260 nm. The concentration of DNA isolated from liver samples (3 g) was 1.9 ± 0.82 mg/g liver. The purity of DNA was determined from absorbance ratios A230/260 and A260/280. 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 (32).
DNA hydrolysis
Calf thymus DNA (500 µg) or rat DNA (200500 µg) was pipetted into a 1.5 ml plastic Eppendorf and spiked with the internal standard dG-C8-4ABP (505 pg). The DNA was sheered by sonication (3 min) in buffer [5 mM BisTris (pH 7.1)/5 mM MgCl2]. The total volume was 350 µl. Deoxyribonuclease 1 (50 µl, 250 U) was added and the reaction was incubated for 3 h, in a shaking water bath, at 37°C. A second buffer [70 µl, 30 mM NaOAc (pH 5.3)/3 mM ZnSO4] and 30 µl of NP1 (9 U) were added and the reaction was incubated for a further 3 h in a shaking water bath at 37°C. Finally, 60 µl of buffer (50 mM TrisHCl, pH 8.8) and 15 µl alkaline phosphatase type III (3 U) were added and the reaction was incubated in a shaking water bath, 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 coupled to a diode array detector (DAD). The HPLC-DAD system consisted of a pump, gradient former, mobile phase degasser and DAD detector (Hewlett Packard series 1100). The dN were separated from 5'P-dN, ribonucleosides and DNA bases using a LiChrospher RP-18 column (2504 mm, 5 µm) with a RP18-pre-column (4x4 mm) at 1 ml/min in mobile phase (94:3:3, 10 mM AF:CH3CN:H2O) and detected at 265 nm wavelength.
Purification and enrichment of DNA adducts
DNA hydrolysates (600 µl), were saturated with NaCl and extracted with EtOAc (1x500 and 2x250 µl), by vortex mixing in 3.5 ml Teflon tubes (Semadeni, CH-3072 Ostermundigen) for 2 min each time. After each extraction the samples were centrifuged for 10 min at 2000 g. The EtOAc phases were combined, spiked with H2O (100 µl), then reduced at room temperature in a speed evaporator (Univapo 150H, UniEquip, D-82512 Martinsried), to
80 µl. The samples were transferred to glass vials (32x12 mm), the recovery standard dG-C8-4CA (100 pg) was added and the total volume was made up to 500 µl. The samples (250 µl) 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 electrospray chemical ionization 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 (125x2 mm, 3 µm) with the following conditions: 5 min isocratic elution with 10% MeOH in 10 mM AF-buffer then a linear 20 min gradient: 10% MeOH in 10 mM AF-buffer to 90% MeOH with a flow rate of 0.25 ml/min.
Synthesis of DNA adducts
The 2'-deoxyadenosine (dA) arylamine adducts were synthesized according to the procedure described in (17). Equivalent molar ratios of N-hydroxy-2MA (60 µmol) and NEt3 (8.4 µl, 60 µmol) were dissolved in THF (3 ml) at -45°C in glass screw capped tubes (100x16 mm) with teflon liners. An equivalent of pyruvonitrile (4.3 µl, 60 µmol) was added to yield the N-acetoxy-2-methylaniline, which was not isolated. The reaction was monitored by thin layer chromatograpy (TLC) (98:2, CHCl3:MeOH) and found to be complete after 90 min. Then dA (15 mg, 52 µmol), in H2O (1 ml) and NEt3 (3.4 µl, 24 µmol) were added and incubated overnight at 37°C. The reaction mixture was evaporated and the residue reconstituted in H2O (5 ml). The aqueous phase was washed with diethyl ether (6x3 ml) and with EtOAc (3x3 ml). The EtOAc extracts were combined and concentrated under vacuum. 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 and 2MA; and from cleavage of the glycosidic bond, respectively.
In vitro reaction of calf thymus DNA with N-acetoxy-2MA
N-acetoxy-2MA solution (-45°C) [synthesized according to ref. (18)] was added drop-wise to a solution of calf thymus DNA (10 mg/1 ml H2O), in EtOH (1.75 ml), CHCl3 (0.75 ml) and NEt3 (3.4 µl) then incubated at 37°C overnight. The mixture was washed six times with equivalent volumes of diethyl ether and EtOAc. The removal of unreacted N-hydroxy-2MA 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 (1000 g), washed with 70% EtOH (1 ml), and then redissolved at
2 mg/ml in H2O. The DNA concentration was determined by UV spectrometry at absorbance 260 nm, assuming that 50 µg DNA/ml yields one absorbance unit at 260 nm. The purity was determined by UV spectrometry at absorbance A230/260 and A260/280.
Hb adducts
Hb was isolated and hydrolyzed as described previously (1820). Hb was precipitated from lysed erythrocytes. The precipitate was washed with ethanol:water (8:2), ethanol, ethanol:diethylether (3:1) and diethylether. To Hb (40 mg) dissolved in 0.1 M NaOH and 0.1% SDS, the internal standard d4-2MA (100 ng) was added. After 1 h gentle shaking at room temperature, the hydrolysate was extracted with hexane (6 ml). The extract was evaporated to 1 ml and 2,4,5-trimethylaniline (245TMA) (100 ng) was added as internal volumetric standard. The analyses were performed on a Hewlett Packard chromatograph (HP 5890II) equipped with an autosampler (HP 7276) 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 0.25 mmx1 m Methyl-Silyl retention gap (Analyt; Müllheim, 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 electron-impact ionization (EI) mode the electron energy was 70 eV and the ion source temperature was 200°C. In the single-ion monitoring (SIM) mode the positive ions m/z = 106 and 107 were monitored for 2MA, m/z = 110 and 111 for d4-2MA, and m/z = 135 for 245TMA. Each of these ions were detected with a dwell time of 50 ms. 2MA eluted with a retention time of tR = 3.78 min, d4-2MA with tR = 3.74 min, and 245TMA with tR = 5.40 min. Helium was used as 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 with d4-2MA (100 ng), 245TMA (100 ng) and 2MA (0200 ng). The recovery of d4-2MA through the experiment was 61 ± 8%.
For structural confirmation the hexane extracts were dried over Na2SO4 and derivatized with pentafluoropropionic anhydride (PFPA) as described in (21). The derivatives were blown to dryness under a gentle stream of nitrogen, taken up in 15 µl hexane and analyzed on the same column described above. 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 negative chemical ionization mode. d4-2MA-PFPA m/z = 237 (100%) and 2MA-PFPA m/z = 233 (100%).
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Results and discussion
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Male WELS-Fohm rats were dosed chronically with 2NT in the drinking water. The animals were killed after 12 weeks. No major toxic effects were noticed in this period. For the present study livers and blood were obtained from three animals per dose.
Determination of Hb adducts
Hb adducts were measured following a protocol used for the analysis of several arylamines (14,15,2023). Deuterated 2MA was used as an internal standard for the analyses. Hb was hydrolysed in NaOH at room temperature. The released 2MA was extracted with hexane and quantified against the deuterated-2MA by GC/MS. For structural confirmation the hexane extracts were derivatized with PFPA and analyzed by GC/MS with negative chemical ionization. From analysis of Hb samples trace levels of 2MA were found in control rats but significantly higher levels were found in rats assigned to each of the exposure groups. The adduct level did increase with the dose as shown in Figure 7. The four different doses (0, 0.29, 0.70 and 1.82 mmol/kg 2NT) yielded 1.2, 20.7, 31.4 and 50.5 pmol 2MA/mg Hb, respectively. In comparison with the low dose, the adduct levels were
1.5 and 2.5-fold lower than expected at the mid and high doses, respectively.

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Fig. 7. Correlation of dose with Hb adducts, dA-2MA, and dG-2MA found in nine exposed rats and three controls.
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In vitro reactions with 2'-deoxynucleosides
According to the current knowledge, the analyzed Hb adducts which are hydrolysable under mild conditions are sulfinamide adducts formed between cysteine residues of Hb, and biologically available N-hydroxy-2MA. Therefore, DNA adducts resulting from the same intermediate were expected. For the analysis of in vivo samples putative reaction products of N-hydroxy-2MA with DNA were synthesized. The dG adduct, dG-C8-2MA [i] had been synthesized previously (18,24). The 2'-deoxyadenosine (dA) adducts were synthesized according to the procedure used for the synthesis of dG adducts. The reaction products of dA with N-hydroxy-2MA were separated by TLC. Each discrete band of silica on the TLC plate was removed and extracted with MeOH. HPLC-MS/MS was used to identify the zone on the TLC plate containing the product from characteristic fragments, [M ± H]± = 357 amu and [BH2]± = 241 amu consistent with formation of a covalent bond between dA and the arylamine; and from cleavage of the glycosidic bond, respectively. The compounds with the correct mass fragments were obtained from the TLC zone with the retention time 0.19. The major products of this extract eluted at 13.4 [ii] and 16.6 min [iv] (Figure 2). A very minor product eluted at 14.8 min [iii].
In vitro reaction with ct-DNA
Experiments were performed to determine whether the N-acetoxy-2-MA, covalently bound to the same positions of dG and dA, within whole ct-DNA, as observed for the reactions between N-acetoxy-2MA and the single bases dG and dA. N-Acetoxy-2MA was reacted with ct-DNA. The DNA was purified by solvent extraction, precipitated and digested enzymatically. The DNA adducts were enriched, by EtOAc, then analyzed by HPLC-ESI-MS/MS. The mass chromatograms for the ions [M ± H]± corresponding to dC, dG, dT and dA-arylamine adducts were monitored. The mass chromatograms for dG and dA-arylamine adducts are presented in Figure 3. For the adducts to dG, the most prevalent peak observed in the MS/MS chromatogram eluted with identical retention times and mass spectra to the peak for dG-C8-2MA (14.1 min) [i], obtained from the single base reaction between dG and N-acetoxy-2-MA (Figures 1
3). In digested ct-DNA from the in vitro modification reactions, an additional peak [v] to the aforementioned dG-C8-arylamine adduct was detected in the selected ion chromatogram at 12.7 min. The MS/MS spectra of these peaks consisted of the same ion fragmentation [M ± H]± and [BH2]±, but a different ratio of m/z 373/257. These ions corresponded to a covalently bound adduct between the pyrimidine base in dG and 2MA. The retention times and MS/MS spectra can be seen in Figures 2 and 3. The exact position of the covalent interaction was not determined because equivalent standards of known structure were not available for comparison. A selection of possible structures for this adduct, which should yield the MS/MS fragmentation pattern observed are shown in Figure 9. When control ct-DNA was digested, extracted and analysed under identical conditions, no peaks were observed in the selected ion chromatograms at retention times corresponding to the dG-C8-2MA [i], or additional dG-2MA adduct [v] found in the modified ct-DNA sample.
The selected ion chromatograms of the in vitro-modified DNA revealed that more than one nucleoside reacted with N-acetoxy-2MA. In addition to the formation of dG arylamine adducts ([i] and [v]), covalent binding to positions on dC (MS/MS m/z 333
217, tR = 14.4 min [vii]), dT (MS/MS m/z 348
232, tR = 14.2 min [viii]) and dA (MS/MS m/z 357
241, tR = 13.3 [ii], 14.9 [iii], 15.6 min [iv]) were identified by HPLC-ESI-MS/MS. For each dN adduct of interest, multiple peaks were observed in the selected ion chromatograms of the digested ct-DNA. With respect to dA-2MA adducts, these peaks ([ii], [iii] and [iv]) 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-acetoxy-2MA.
Determination of DNA adducts in rats dosed chronically with 2NT
At the end of the treatment regime the rats were killed and their livers were excised. Hepatic DNA from each dose group of rats (N = 3) was precipitated and purified from whole liver. Hepatic DNA was spiked with internal standard, dG-C8-4ABP, then sequentially digested to individual 2'-deoxyribonucleosides (dN) with DNase 1, NP1 and AP. For an accurate assessment of DNA hydrolysis, by enzymatic digestion, quantification of each dN (dC, dG, dT and dA) released in the DNA digest was performed prior to analysis of DNA adducts by HPLC-ESI-MS/MS. Under selected chromatographic conditions, the dN eluted independent of each other and separately from the other nucleic acid constituents (Figure 6). The mole percent ratios of dN pairs, dC with dG and dT with dA, determined from 100 to 500 µg of digested ct-DNA lay within the literature range: GC = 41.9 and AT = 58.1 (25).

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Fig. 6. HPLC coupled with DAD detection. Constituents of nucleic acid were separated isocratically, on a LiChrospher RP-C18 column (2504 mm, 5 µm), at 1 ml/min in mobile phase (94:3:3, 10 mM AF, MeOH, AcCN, pH 4.0) and their absorbance (mAU) was detected at 265 nm. Panel 1: Standard solution (1 mg/10 ml) of individual dNs, dC, dG, dT and dA, and analogous 5'-monophosphate dNs, dC-5'P, dG-5'P and dA-5'P. The retention time and peak assignment have been denoted above each peak. Under the same conditions: the bases G, C, T and A elute at 2.7, 3.7, 5.1 and 6.1 min, respectively. The ribonucleosides rC, rG, rU and rA elute at 3.2, 3.8, 7.3 and 16.3 min, respectively.
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Modified dN were enriched by liquid partitioning into EtOAc, evaporated, reconstituted in H2O containing the recovery standard dG-C8-4CA and analyzed by HPLC-MS/MS (Figure 4). For quantification of DNA adducts the [M ± H]± of dG-2MA (m/z 373) and dA-2MA (m/z 357) were selected and CID was initiated by application of 20% CE. One major adduct to dG (Figure 4 [vi]) and one major adduct to dA (Figure 4 [iii]) were found. The predominant ions formed due to CID of the dG-2MA adduct [vi] were [M ± H]±, and daughter ions, [M ± H - 116]± and [M ± H - 18]±. The [M ± H - 116]± ion was consistent with the loss of the deoxyribose sugar, resulting from cleavage of the glycosidic bond. The [M ± H - 18]± ion corresponded to loss of H2O. An additional minor ion, [M ± H - 17]±, was tentatively characterized as loss of the guanine NH2 group and the guanine N1 hydrogen. The predominant ions formed due to CID of the dA-2MA adduct [iii] were the [M ± H]± and daughter ion, [M ± H - 116]±. The [M ± H - 116]± ion was consistent with the loss of the deoxyribose sugar, resulting from cleavage of the glycosidic bond. An additional minor ion, [M ± H - 18]±, was characterized as loss of H2O.
The retention time of the dG-2MA adduct [vi], found in chronically dosed rats (N = 3), ranged between 12.3 and 12.5 min. The drift in retention time disappeared when the peak was related to the retention time of the recovery standard, dG-C8-4CA. The dG-2MA adduct [vi] detected in the exposed rats did not have the same MS/MS fragmentation pattern as the structurally resolved dG-C8-2MA standard [i], and eluted 1.6 min beforehand (Figures 2 and 4). The dA-2MA adduct [iii] detected in the exposed rats (Figure 4) possessed the same MS/MS fragmentation and retention time as seen for the minor peak identified in the reaction between dA and N-acetoxy-2MA [iii] (Figure 2).
The dG-2MA adduct discovered in hepatic DNA, from dosed rats, had the same retention time as one of the adducts present in ct-DNA, modified in vitro with N-acetoxy-2MA (Figure 3). There were two peaks in the selected ion chromatogram of the digested in vitro-modified ct-DNA, which corresponded to dG-2MA adducts. The major dG-2MA adduct, which eluted at 14.1 min was identified as the dG-C8-2MA [i] adduct by comparison with the structurally resolved standard (Figure 3). The second peak [v], which eluted prior to the dG-C8-2MA adduct, at 12.7 min, had an identical retention time (when related to the recovery standard dG-C8-4CA) to the unknown dG-2MA adduct [vi] detected in digested hepatic DNA from dosed rats, but a different MS/MS fragmentation. The in vivo dG-2MA adduct showed an additional major (M-18) fragment. This highlights one of the advantages MS/MS confers over other analytical tools, which may not have been able to discriminate between the two different adducts with the same retention time.
In in vitro-modified DNA, three peaks corresponded to dA-2MA adducts. The minor dA-2MA adduct, observed in the selected ion chromatogram of the single base reaction between dA and N-acetoxy-2MA (Figure 2, [iii]), and the in vitro modification reaction between ct-DNA and N-acetoxy-2MA (Figure 3, [iii]), eluted at an identical retention time (14.9 min, [iii]) to the dA-2MA adduct observed in digested hepatic DNA, from dosed rats (Figure 4, [iii]). The major peaks, at 13.3 min [ii] and 16.5 min [iv], in the single ion chromatograms of the single base reaction between dA and N-acetoxy-2MA (Figure 2), and the in vitro modification reaction between ct-DNA and N-acetoxy-2MA (Figure 3), were not found in the chronically dosed rats.
The level of the dG-2MA adducts found in the hepatic DNA was estimated from a calibration line obtained from dG-C8-2MA. As the structurally unknown dG-2MA adduct [vi] was not available for precise quantification, the dG-C8-2MA standard was used. Different amounts (0, 20, 100 and 500 pg) of dG-C8-2MA were spiked into ct-DNA (500 µg) with internal standard, dG-C8-4ABP (508 pg), and taken through the work up procedure and analyzed by HPLC-ESI-MS/MS as described for the 2NT chronically dosed rat samples. Each data point in the calibration line (y = 0.0293x ± 0.0798, r2 = 0.995) of dG-C8-2MA was an averaged value ± the standard deviation calculated from six independent determinations.
The overall recovery of the procedure was determined with dG-C8-2MA (20 or 100 pg) spiked with internal standard dG-C8-4ABP (508 pg) into digested ct-DNA (500 µg). The samples were extracted and treated as the in vivo samples. The recovery standard dG-C8-26DMA (100 pg) was added prior HPLC-MS/MS analysis. The recovery of 20 and 100 pg dG-C8-2MA were 26 ± 3.6% (± relative standard deviation) and 28 ± 12%, respectively.
No peak, corresponding to a dG-2MA adduct, was observed in the selected ion chromatogram of digested control rat DNA (Figure 5). There was a peak in the selected ion chromatogram of digested control rat DNA, which corresponded to an adduct to dA (Figure 5). This peak had an identical retention time but different MS/MS fragmentation pattern to that seen for the 2NT-dosed rats. The integrated peak area of the dA-2MA adduct in the control rats was 100-fold less than the peak observed in the lowest 2NT-dosed group (0.29 mmol/kg). Very low levels of Hb adducts were found also in the control rats of this experiment (see above). Therefore, it appears that also the control rats were exposed to low levels of 2NT, as in control rats (female Wistar rats) not involved in this experiment no peak for dA-2MA was detected.

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Fig. 5. HPLC/MS/MS chromatogram of digested hepatic DNA from a control rat. HPLC/MS/MS conditions see legend to Figure 4. The peak area of the dA-2MA adduct in the control rats was 100-fold less than the peak observed in the lowest 2NT-dosed group.
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The concentration of dG-2MA was plotted against the dose of 2NT administered to the rats (mmol/kg/day). The plot presented in Figure 7 showed that there was a linear doseresponse relationship between dG-2MA adduct levels, and the administered dose of 2NT, over the range of 0, 0.29 and 0.70 mmol/kg. The level (average ± standard deviation = 3.53 ± 0.5 pmol/mg DNA) of dG-2MA adducts in the highest dose group deviated from linearity. The level was 41% below the predicted level (= 5.96 pmol/mg DNA) determined by the regression line (y = 3.269x ± 0.0132) through data points for 0, 0.29 and 0.70 mmol/kg/day. The level of dG-2MA adducts determined in these rats ranged between 0 and 4.1 pmol/mg hepatic DNA. The data were also expressed in terms of a modification level, given as the number of adducts per 107 nucleotides. In the lowest 2NT-dosed group (0.29 mmol/kg) the average modification level ± the standard deviation (N = 3) was 3.06 ± 1.02 per 107 nucleotides. For 0.70 and 1.82 mmol/kg the modification levels were 7.09 ± 1.50 per 107 and 10.92 ± 1.56 per 107 nucleotides, respectively. These adduct levels are comparable with the levels found in livers of rats given 2-nitrofluorene (0.156 mmol/kg/10 days) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.44 mmol/kg/10 days): 5.4 per 107 (27) and 5.0 per 107 (278) nucleotides, respectively.
dA-2MA was quantified with the same calibration curve as dG-2MA. The doseresponse relationship for the dA-2MA adducts was similar as for the dG-2MA adducts (Figure 7). The level of dA-2MA adducts in the highest dose group (averaged ratio of dA-2MA = 20.1 ± 4.45 pmol/mg DNA) deviated from linearity. The level was 39% below the adduct level (dA-2MA = 32.93 pmol/mg DNA) predicted from the regression line (y = 18.16x - 0.128) of the linear doseresponse curve over 0, 0.29 and 0.70 mmol/kg.
The most plausible explanation for such a supralinear doseresponse relationship in rats chronically administered 2NT, was that saturation of metabolic activation processes had occurred at the highest administered dose, which resulted in a non-linear increase in the apparent levels of DNA and Hb adducts. A similar dose response relationship was found for liver DNA adducts in rats dosed 4-aminobiphenyl (26). There is an excellent linear correlation between the DNA adducts dG-2MA and dA-2MA; r2 = 0.90 (Figure 8A). In addition the Hb adducts correlate with the dG-2MA and the dA-2MA adducts with r2 = 0.90 and 0.91, respectively (Figure 8B).

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Fig. 8. (A) Correlation of the DNA adducts: dG-2MA versus dA-2MA: y = 5.44x ± 0.15, r2 = 0.90. (B) Correlation of the DNA adducts with Hb adducts: dG-2MA versus Hb adducts: y = 12.6x ± 4.6, r2 = 0.90. dA-2MA versus Hb adducts: y = 2.2x ± 5.2, r2 = 0.91.
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The animal data were compared with preliminary human data obtained in our laboratory. We examined a group of workers exposed to an average of 0.74 mg 2NT/m3 air, a level which is 40 times below the permissible daily levels set by the Occupational Safety and Health Administration (6). The mean level of 2MA-Hb adducts in the exposed workers, excluding 2 outliers (26.9 and 88.6 fmol/mg Hb) was 6.5 ± 3.3 fmol/mg Hb. This 2MA-Hb adduct level was
3200 times below the 2MA-Hb adduct levels determined in the low-dosed rats. Assuming that in humans we have the same dose responses as in rats, the 2MA-dG adduct levels would be 1 adduct/1010 nucleotides, which is below the detection limit of our method, and which is below the levels of arylamine adducts found in other human studies with more potent carcinogens like 4-aminobiphenyl (1100 adducts/109 nucleotides) (26).
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Conclusions
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In rats dosed 2NT Hb- and DNA adducts were found. The dG and dA adducts found in the 2NT-dosed rats increased with the dose. The same increase was seen for the Hb adduct levels measured in the same animals. The increase of DNA and Hb adduct levels were supralinear. A strong linear relationship also existed between the level of dG2MA or dA2MA adducts, in hepatic DNA, and Hb adducts, over the whole dose range (r2
0.9).
The hydrolyzable Hb adducts result from the activation of the nitro-group. The DNA adducts could result from the activation of the nitro-group or from the activation of the methyl group, as the structure could not be elucidated. The expected adduct with C8-of dG was not found. In the future a large number of adducts have to be synthesized in order to characterize the DNA adducts found in vivo. Several adducts are possible with dG following the activation of the nitro-group (Figure 9) (29). In addition, the adducts resulting from the activation of the methyl-group should be investigated. The activated methyl-group of 2NT reacts with the same centers as the nitrenium ion (Figures 1 and 9). The same applies to the adducts with dA. N2 and N6 adducts have been synthesized recently (30,31). The C8 adducts of adenine with 2MA are presently unknown.
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Acknowledgments
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We acknowledge the technical assistance of Renate Hartley and the financial support by the European Commission, ERB-IC-CT97-0221.
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References
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Received July 8, 2002;
revised November 26, 2002;
accepted December 3, 2002.