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Carcinogenic 3-nitrobenzanthrone but not 2-nitrobenzanthrone is metabolised to an unusual mercapturic acid in rats
Toxicology Letters 208 (2012) 246–253
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Carcinogenic 3-nitrobenzanthrone but not 2-nitrobenzanthrone is metabolised to an unusual mercapturic acid in rats a ˇ Igor Linhart a,∗ , Jaroslav Mráz b , Iveta Hanzlíková b , Alexandra Silhánková , Emil Frantík b , Michal Himl a a b
Department of Organic Chemistry, Faculty of Chemical Technology, Institute of Chemical Technology, Prague, Technická 1905, CZ-166 28 Prague, Czech Republic National Institute of Public Health, Sˇ robárova 48, CZ-100 42 Prague, Czech Republic
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 18 November 2011 Accepted 19 November 2011 Available online 26 November 2011 Keywords: Nitrobenzanthrones Environmental carcinogens Biotransformation Urinary metabolites Mercapturic acid
a b s t r a c t 3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspect human carcinogen found in diesel exhaust. Its isomer 2-nitrobenzanthrone (2-NBA) has also been found in ambient air. These isomers differ in mutagenicity in Salmonella by 2–3 orders of magnitude. To identify their urinary metabolites and also to assess the assumed differences in their excretion, rats were dosed orally with 2 mg/kg b.w. of either 2-NBA or 3-NBA. Their urine was collected for two consecutive days after dosage. Both LC–ESI-MS and GC–MS confirmed formation of the corresponding aminobenzanthrones (ABA). Excretion of these metabolites within the first day after dosing with 2- and 3-ABA amounted to 0.32 ± 0.06 and 0.83 ± 0.40% of the doses, respectively, while the excretion within the second day was by one order of magnitude lower. A novel mercapturic acid metabolite of 3-NBA was identified in urine by LC–ESI-MS as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABA-MA) by comparison with the authentic standard. Its excretion amounted to 0.49 ± 0.15 and 0.02 ± 0.01% of dose within the first and second day after dosing, respectively. In contrast, no mercapturic acid was detected in the urine of rats dosed with 2-NBA. Observed difference in the mercapturic acid formation between 2and 3-NBA is a new distinctive feature reflecting differences in the critical step of their metabolism, i.e., benzanthronylnitrenium ion formation that is intrinsically associated with biological activities of these two isomers. Moreover, 3-ABA-MA is a promising candidate biomarker of exposure to the carcinogenic 3-NBA. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Both 2- and 3-nitro-7H-benz[de]anthracen-7-one (2- and 3nitrobenzanthrone, NBA), like numerous other polycyclic nitroaromatics, are important environmental pollutants emitted from diesel engines. They are found mainly adsorbed on the surface of airborne particulate matter and are of great concern due to their mutagenic and carcinogenic potential (Enya et al., 1997; Arlt, 2005; Arlt et al., 2007). Although both are mutagenic in Salmonella typhimurium striking differences exist in their mutagenic potential with 3-NBA being by 2–3 orders of magnitude more mutagenic than the 2-nitro isomer (Takamura-Enya et al., 2006). Number of revertants induced in the Ames test by 3-NBA is comparable to that of 1,8-dinitropyrene, one of the most potent mutagens known (Purohit and Basu, 2000; Arlt, 2005). Carcinogenicity of 3-NBA was demonstrated in an experiment on rats following intratracheal instillation (Nagy et al., 2005). Numerous 32 P-postlabelling and MS studies have shown formation of DNA adducts in rats dosed with
∗ Corresponding author. Tel.: +420 220 444 165; fax: +420 220 444 288. E-mail address: [email protected] (I. Linhart). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.11.017
3-NBA as well as in experiments with metabolically activated 3NBA in vitro (Bieler et al., 2005, 2007; Arlt et al., 2001, 2006; da Costa et al., 2009). DNA adducts were formed also from 2-NBA in rat lungs in vivo, although only at very high doses of 2-NBA (Arlt et al., 2007). 3-NBA is metabolically reduced to 3-(hydroxyamino) benzanthrone (3-OH-ABA), which is either activated by Oacetylation and sulphate conjugation or further reduced to 3-aminobenzanthrone (3-ABA). 3-ABA was identified in the urine of mining workers heavily exposed to diesel emissions (Seidel et al., 2002). Both acetate and sulphate conjugates dissociate to electrophilic 3-benzanthronylnitrenium ion, which is the ultimate DNA reactive species (Scheme 1). Reduction of 3-NBA is catalysed mainly by NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) (see Scheme 2). In general, electrophilic species are detoxified by mercapturic acid pathway (MAP) initiated by conjugation with glutathione (GSH), followed by subsequent cleavage of glutamine and glycine, and terminated by acetylation to yield S-substituted N-acetylcysteines, mercapturic acids, which are excreted in urine. Mercapturic acids can serve as valuable biomarkers of exposure reflecting electrophilic reactivity of xenobiotics and/or
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Scheme 1. Preparation of the mercapturic acids derived from 2- and 3-NBA by reaction of the corresponding activated OH-ABAs with NAC.
their metabolites (for reviews see Haufroid and Lison, 2005; Angerer et al., 2007). Numerous in vitro studies on the metabolic activation of 3-NBA were published (Borlak et al., 2000; Arlt, 2005; Hansen et al., 2007; Stiborová et al., 2006), however, only one urinary metabolite, 3aminobenzanthrone, has been identified as yet (Seidel et al., 2002). Arylation of N-acetylcysteine (NAC) and GSH by activated hydroxyaminoarenes (Boyland et al., 1962; Manson, 1972; Ketterer et al., 1979) as well as by benzidine upon oxidative activation by horseradish peroxidase (Josephy and Iwaniw, 1985) has been described long ago. Therefore, it is rather surprising that, to our knowledge, very few mercapturic acids formed via arylnitrenium ions in vivo have been reported so far, namely, 2and 4-aminophenylmercapturic acids derived from aniline and 2amino-1-naphthylmercapturic acid derived from 2-naphthylamine (Boyland et al., 1963). The aim of the present work was to identify urinary metabolites of both 2- and 3-NBA, in particular those produced through the formation of nitrenium ion intermediates. The amounts of mercapturic acids possibly found after administration of 2- and 3-NBA would reflect the extent of formation of the corresponding benzanthronylnitrenium ions and, indirectly, the extent of toxic insult caused by each NBA isomer.
2. Materials and methods 2.1. Materials Acetonitrile for LC/MS Chromasolv was from Merck, formic acid, puriss. p.a. from Fluka. Re-distilled water was used for LC/MS and solid phase extraction. Tetrahydrofuran (THF) was dried by distillation from sodium and benzophenone. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene bound to polystyrene (TBD-methyl polystyrene) was from Novabiochem, Germany. Heptafluorobutyric anhydride (HFBA) was from Fluka. Other chemicals were of synthetic or analytical grade and were used as received.
2.2. Syntheses of nitrobenzanthrones and their expected metabolites 2-Nitrobenzanthrone (2-NBA) was prepared by a published multi-step synthesis (Suzuki et al., 1997) with a modification in the key step. Instead of Ullmann reaction, which gave low yields of the key intermediate 1-(2methoxycarbonylphenyl)-3-nitronaphthalene, Suzuki coupling (Miyaura et al., 1979) of 1-iodo-3-nitronaphthalene with 2-(methoxycarbonyl)phenylboronic acid was used.
3-Nitrobenzanthrone (3-NBA) was obtained by direct nitration of benzanthrone with fuming nitric acid as described in the literature (Enya et al., 1998). 2-Aminobenzanthrone (2-ABA). Palladium catalyst (10 mg of 10% Pd on charcoal, Degussa type containing ca. 50% of water) was activated by heating at 90 ◦ C for 1 h in a vacuum. Argon was then introduced into the reaction flask and 2-NBA (15 mg, 54.5 mol), diglym (30 mL) and hydrazine hydrate (75 L, 77 g, 1.54 mol) were added. The reaction mixture was stirred at room temperature under argon and the course of reaction was monitored by HPLC with a PDA detector. After 2 days conversion of 2-NBA was complete. The catalyst was then filtered off and the filtrate was evaporated in a vacuum to dryness. Crystallisation of the residue from ethanol yielded 9.5 mg (71%) of orange crystals identified by comparing their 1 H NMR spectrum with that reported in the literature as 2-ABA (Takekawa et al., 2002). ESI-MS: m/z 246 (M+H)+ , 268 (M+Na)+ . MS2 : m/z 246 → 228 (MH−H2 O)+ , 229 (MH−NH3 )+ ; 230 (MH−O)+ ; 217 (MH−COH)+ ; 218 (MH−CO)+ , 219 (MH−C2 H2 )+ , 201 (MH−CONH3 )+ . UV: max = 442, 375, 303 and 230 nm. 2-Acetamidobenzanthrone (N-acetyl-2-aminobenzanthrone, 2-AcABA). 2-ABA (10 mg, 41 mol) was refluxed in acetic anhydride (1.5 mL) for 4 h. Reaction mixture was then evaporated to dryness in a vacuum. Re-crystallisation of the residue from aqueous ethanol yielded 6.5 mg (56%) of greenish-yellow crystals. 1 H NMR spectrum (CDCl3 ): ı = 2.40 (s, 3H, CH3 CO); 7.61 (td, J = 7.5 and 1.0 Hz, 1H, C9-H); 7.76 (td, J = 7.7 and 1.5 Hz, 1H, C10-H); 7.82 (d, J = 1.8 Hz, 1H, C3-H); 7.84 (dd, J = 8.2 and 7.6 Hz, 1H, C5-H); 8.19 (d, J = 1.9 Hz, 1H, C1-H); 8.22 (dd, J = 8.2 and 1.2 Hz, 1H, C11-H); 8.28 (d, J = 8.2 Hz, 1H, C4-H); 8.52 (dd, J = 7.9 and 1.1 Hz, 1H, C8-H); 8.80 (dd, J = 7.3 and 1.4 Hz, 1H, C6-H). ESI-MS: m/z 288 (M+H)+ , MS2 : m/z 288 → 246 (MH−CH2 CO)+ . UV: max = 389, 307, 256 and 240 nm. 2-(Hydroxyamino)benzanthrone (2-OH-ABA). To a suspension of 2-NBA (15 mg, 54.5 mol) in diglym (30 mL) palladium catalyst (15 mg of 5% Pd on charcoal) and hydrazine hydrate (75 L, 77 g, 1.54 mol) was added. The reaction mixture was then stirred overnight under argon at the room temperature. Conversion of 2-NBA amounted to more than 90% as determined by HPLC with PDA detector. After filtering off the catalyst, the solvent was evaporated in a vacuum to dryness, and re-suspended in 20 mL of a 1:1 ethyl acetate–chloroform mixture. The solid was filtered off to yield 5 mg (35%) of a greenish yellow powder identified as 2-OH-ABA. 1 H NMR spectrum (DMSO-d6 ): ı = 7.51 (bs, 1H, NH); 7.64 (t, J = 7.5 Hz, 1H, C9-H); 7.75 (t, J = 7.8 Hz, 1H, C10-H); 7.86 (t, J = 7 Hz, 1H, C5-H); 8.22 (d, J = 2 Hz, 1H, C3-H); 8.25 (d, J = 7.5 Hz, 1H, C4-H); 8.32 and 8.35 (d, J = 6.7 Hz, 2H, C6-H and C8-H); 8.41 (d, J = 8.2 Hz, 1H, C11-H); 8.78 (d, J = 1.8 Hz, 1H, C1-H); 8.88 (bs, 1H, OH). ESI-MS: m/z 262 (M+H)+ ; MS2 : 262 → 245 (MH−NH3 )+ , 244 (MH−H2 O)+ , 234 (MH−CO)+ , 217 (MH−CO−NH3 )+ . UV: max = 430, 375, 297 and 224 nm. 3-Aminobenzanthrone (3-ABA). 3-NBA (20 mg, 72.7 mol) was dissolved in 20 mL of diglym, palladium catalyst (20 mg of 5% Pd on charcoal) and hydrazine hydrate (80 L, 82 mg, 1.65 mol) was added and the reaction mixture was stirred overnight under argon at room temperature. The catalyst was then filtered off, the filtrate was evaporated in a vacuum to dryness and the residue re-dissolved in 5 mL of ethanol. Red precipitate formed after dilution of this solution with water was filtered off and dried over P4 O10 . Dark red powder of 3-ABA obtained (9 mg, 51%) showed 1 H NMR spectrum, which was in agreement with published data (Safronov and Traven, 1993).
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Scheme 2. Biotransformation of 2- and 3-NBA to the urinary metabolites found in rats in vivo. NQO1 – NAD(P)H:quinone oxidoreductase 1, NAT – N-acetyltransferase, SULT – sulfotransferase; MAP – mercapturic acid pathway.
ESI-MS: m/z 246 (M+H)+ , 268 (M+Na)+ . MS2 : m/z 246 → 228 (MH−H2 O)+ , 229 (MH−NH3 )+ ; 230 (MH−O)+ ; 217 (MH−COH)+ ; 218 (MH−CO)+ , 219 (MH−C2 H2 )+ , 201 (MH−CONH3 )+ . ESI-MS: m/z 246 (M+H)+ ; 268 (M+Na)+ . UV: max = 507, 280 and 218 nm. 3-Acetamidobenzanthrone (N-acetyl-3-aminobenzanthrone, 3-AcABA) was obtained as described for 2-AcABA as yellow-brown crystals from aqueous ethanol. 1 H NMR spectrum (CDCl3 ): ı = 2.37 (s, 3H, CH3 CO); 7.56 (d, J = 7.9 Hz, 1H, C2-H); 7.6 (t, J = 8 Hz, 1H, C9-H); 7.78 (td, J = 7.8 and 1.5 Hz, 1H, C5-H); 7.85 (t, J = 8 Hz, 1H, C10-H); 8.07 (dd, J = 8 and 1.2 Hz, 1H, C11-H); 8.34 (d, J = 8.2 Hz, 1H, C1-H); 8.50 (dd, J = 8 and 1.2 Hz, 1H, C8-H); 8.52 (d, J = 7.8 Hz, 1H, C6-H); 8.80 (dd, J = 7.5 and 1.2 Hz, 1H, C4-H). ESI-MS: m/z 288 (M+H)+ , MS2 : m/z 288 → 246 (MH−CH2 CO)+ . UV: max = 416, 310, 262 and 237 nm.
3-(Hydroxyamino)benzanthrone (3-OH-ABA) was prepared by reduction of 3-NBA with hydrogen on palladium as described earlier (Osborne et al., 2005). (3-aminobenzanthron-2N-Acetyl-S-(3-aminobenzanthron-2-yl)cysteine ylmercapturic acid, 3-ABA-MA). This mercapturic acid was obtained by the reaction of 3-(acetoxyamino)-benzanthrone (3-AcO-ABA) with NAC. 3-AcO-ABA was prepared by reacting 25 mg (96 mol) of 3-OH-ABA with pyruvonitrile in presence of TBD-methyl polystyrene as published previously (Arlt et al., 2006). Then, its solution in 20 mL of TFH was added immediately to 5 mL of aqueous solution of NAC (19 mg, 117 mol) pH of which was adjusted to 7.6. The reaction mixture was left standing overnight at the room temperature, thereafter diluted with chloroform (30 mL) and extracted with 20 mL of 0.1 M Na2 HPO4 (pH 9.1). Aqueous solution was acidified to pH 3.2 with 1 M HCl and extracted twice with 10 mL portions of ethyl acetate. Evaporation of the ethyl acetate extract to dryness in a vacuum yielded 2 mg of crude product. Combined crude products from two
I. Linhart et al. / Toxicology Letters 208 (2012) 246–253 experiments were purified by semi-preparative HPLC on a 250 × 10 mm I.D. column Phenomenex Luna C18(2), 10 m particle size. The column was eluted at a flow rate of 2.8 mL/min with a gradient of acetonitrile in aqueous formic acid (0.15%). Concentration of acetonitrile increased linearly from 35.5% to 50% within 20 min. The peak of 3-ABA-MA was collected at 17.8 min. 1 H NMR (acetone-d6 ): ı = 1.90 (s, 3H, CH3 CO); 3.25 (dd, J = 7.6 and 14 Hz, 1H, CH2 S); 3.41 (dd, J = 4.1 and 14 Hz, 1H, CH2 S); 4.75 (m, 1H, CHN); 6.92 (bs, 2H, NH2 ); 7.42 (t, J = 7.6 Hz, 1H, C10-H); 7.76 (t, J = 7.8 Hz, 1H, C5-H); 7.83 (t, J = 7.2, 1H, C9-H); 8.37 (dd, J = 1.5 and 7.6 Hz, 1H, C11-H); 8.51 (d, J = 7.9 Hz, 1H, C8-H); 8.74 (d, J = 7.3 Hz, 1H, C4-H or C6-H); 8.76 (d, J = 8 Hz, 1H, C4-H or C6-H); 8.78 (s, 1H, C1-H); 7.57 (bd, J = 7.5 Hz, 1H, NH). ESI-MS: m/z 407 (M+H)+ , 405 (M−H)− ; MS2 : 407 → 365 (MH−CH2 CO)+ , 278 (MH−CH2 C(NHCOCH3 )COOH)+ , 246 (MH−SCH2 C(NHCOCH3 )COOH)+ , 405 → 276 (M−SCH2 CH(NHCOCH3 )COO)− ; HRMS: calculated for C22 H19 N2 O4 S+ 407.10600; found 407.10587. UV: max = 504, 288 and 226 nm. Reaction of 2-(acetoxyamino)benzanthrone (2-AcO-ABA) with N-acetylcysteine. THF solution (12 mL) of 2-AcO-ABA that was prepared from 15 mg (57 mol) of 2-OH-ABA as described above for 3-AcO-ABA, was immediately added to a solution of 11 mg (66 mol) of NAC in 3 mL of water (pH 7.6). The mercapturic acid formed was isolated as described above for 3-ABA-MA. Its amount was not sufficient for full structure characterisation, however, fragmentation in ESI-MS2 of both (M+H)+ and (M−H)− afforded the same ions as 3-ABA-MA, thus suggesting strongly that the product is 2-ABA-MA. ESI-MS: m/z 407 (M+H)+ , 405 (M−H)− ; MS2 : 407 → 365 (MH−CH2 CO)+ , 278 (MH−CH2 C(NHCOCH3 )COOH)+ , 246 (MH−SCH2 C(NHCOCH3 )COOH)+ , 405 → 276 (M−SCH2 CH(NHCOCH3 )COO)− . UV: max = 451, 377, 314, 235 nm.
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another 5 min. The flow rate was 200 L/min. Mass spectra (positive ions) were taken in the range of m/z 150–450. MS2 spectra were taken at m/z 246 for 2- and 3-ABA with a collision energy of 36 V and at m/z 288 for 2- and 3-Ac-ABA with a collision energy of 25 V. Transitions at m/z 246 → 219, 246 → 218 and 246 → 217 were used for quantification of 2-and 3-ABA, transition at m/z 288 → 246 for quantification of 2- and 3-ABA. Calibration samples were prepared by spiking blank urine to provide analyte concentrations from 1.5 to 30 ng/mL for 2- and 3-ABA and from 3 to 30 ng/mL for 2- and 3-AcABA. The calibration curves were linear (R2 > 0.99). 2.5.2. Acidic metabolites Urine samples were neutralised to pH 7.0–7.5 with 0.1 M HCl and filtered through 0.45 m Nylon membrane filters. Filtrates (1 mL aliquots) were diluted with 2 mL of water and poured onto pre-conditioned 60 mg Oasis HLB SPE columns (Waters GmbH). The columns were washed with 3 mL of water followed by 3 mL of 20% aqueous methanol and finally eluted with 3 mL of methanol. The eluates were evaporated to dryness at 40 ◦ C in the stream of nitrogen, residues were re-dissolved in 300 L of 40% acetonitrile for LC–ESI-MS analysis. Aliquots (10 L) were injected onto a BDS Hypersil C18 column (150 × 2 mm I.D., 5 m particle size; Thermo). The column was eluted with a gradient of acetonitrile in 0.075% aqueous formic acid. Concentration of acetonitrile was increased linearly from 9% to 65% within 20 min and than to 83% within another 2 min. Mass spectra (positive ions) were taken in the range of m/z 150–500. MS2 spectra were taken at m/z 407 for ABA-MA with a collision energy of 32 V. Additionally, negative ion spectra at m/z 405 with a collision energy of 21 V were taken in a separate run. Transition at m/z 407 → 278 in the positive ion mode was used for quantification of 3-ABA-MA. Calibration samples were prepared by spiking blank urine to provide analyte concentrations from 5.2 to 208 ng/mL. The calibration curve was linear in this range of concentrations (R2 > 0.99).
2.3. Analytical instrumentation LC/MS analyses were carried out on a Thermo Scientific LXQ linear trap mass spectrometer in tandem with a Janeiro LC system consisting of two Rheos 2200 pumps and CTC PAL autosampler. ESI in both positive and negative ion modes was used. Ion source capillary temperature was set to 300 ◦ C. In some experiments a Thermo Scientific Surveyor PDA detector was used instead of or in series with the MS detector. NMR spectra were taken on a Varian Gemini 300 MHz spectrometer. High resolution MS was measured on a Thermo Scientific LTQ Orbitrap Velos spectrometer. GC/MS analyses were performed on a 7890 gas chromatograph equipped with 5975C inert XL EI/CI MSD (Agilent). Samples were introduced onto a capillary column DB-5ms, 30 m × 0.25 mm I.D. with 0.25 m film thickness (Agilent). Injector was set in a splitless injection mode. The column oven temperature was held at 80 ◦ C for 1 min, then raised at 30 ◦ C/min to 320 ◦ C and held at this temperature for 5 min. Injector temperature was 250 ◦ C. Helium was used as the carrier gas at a linear velocity of 30 cm/s. MS detector was operated in the electron ionisation (EI) mode at 70 eV, and was set in a SIM mode at m/z 441 for determination of HFB derivatives of ABA. 2.4. Animal treatment Adult male Wistar rats, average weight 276 ± 6 g (mean ± SD, n = 8) were placed individually into glass metabolic cages with free access to pelleted food and water. To enhance diuresis, sucrose (8 mg/mL) was added to the drinking water. Control urine was collected for 24 h. 2-NBA and 3-NBA were dissolved in DMSO at concentrations of 0.5 and 1 mg/mL, respectively. Concentration of these solutions was limited by solubility. Three rats were dosed by gavage with 4 mL/kg of the 2-NBA solution, another three rats were dosed with 2 mL/kg of the 3-NBA solution, so that both groups received a dose of 2 mg/kg of one NBA isomer. Two control animals were dosed with DMSO only. Urine was collected in the time intervals from 0 to 24 h and from 24 to 48 h after administration. During sample collection the urine was filtered through a gauze filter to remove pieces of faeces and crumbs of food pellets. The walls of the metabolic cages were rinsed with distilled water and resulting solution was added to the main portion of collected urine. Samples were stored at −20 ◦ C until analysed. Animal experiments were approved by the Commission for Animal Protection of the NIPH and of the Czech Ministry of Health. 2.5. Analyses of urine by LC–ESI-MS 2.5.1. Non-acidic metabolites Urine samples were neutralised to pH 7.5–7.8 with 0.1 M HCl and filtered through 0.2 m Nylon membrane filters. Filtrates (1 mL aliquots) were diluted with 2 mL of water and poured onto 60 mg Oasis HLB SPE columns (Waters GmbH), which were pre-conditioned by washing them with 3 mL of methanol followed by 3 mL of water. The columns were washed subsequently with 3 mL of 10% aqueous methanol and 3 mL of 50% methanol and eluted with 3 mL of 1:1 mixture of methanol and acetonitrile. The eluates were evaporated to dryness at 40 ◦ C in the stream of nitrogen and the residues were re-dissolved in 300 L of 40% acetonitrile for LC–ESI-MS analysis. Aliquots (10 L) of the urine extracts were injected onto a BDS Hypersil C18 column (150 × 2.1 mm I.D., 5 m particle size). The column was eluted with a gradient of acetonitrile in 0.075% aqueous formic acid. Concentration of acetonitrile increased linearly from 9% to 83% within 20 min and was then kept constant for
2.6. Determination of total 2- and 3-ABA in urine by GC–MS Urine was analysed for total (free plus conjugated) 2- or 3-ABA by a modified method as described in Lind et al. (1997). Urine samples (1 mL) were acidified with 1 mL of 3 M sulphuric acid the mixture was then vortexed in a 10 mL screw-cup tube and heated at 100 ◦ C for 2 h. After cooling, 10 M NaOH (2 mL) was added and the alkalinised sample was extracted with toluene (2 mL). The organic layer was separated, HFBA (20 L) was added and resulting mixture was heated at 50 ◦ C for 1 h. The organic layer was washed with 0.1 M phosphate buffer pH 7 (1.5 mL) and then taken in a vacuum concentrator to dryness. The residue was taken up in toluene (100 L) and analysed by GC/MS. Quantification of ABA in urine was carried out using standard addition method. Thus, with each sample, another aliquot was worked-up in parallel, to which aqueous solution of the respective ABA (1 g/mL, 100 L) was added.
3. Results 3.1. Preparation of authentic standards Reduction of 2- and 3-NBA by hydrogen on palladium yielded corresponding OH-ABA and, when the reaction time was prolonged, ABA as the end-product. The two NBA isomers significantly differed in their reactivity. Conversion of 3-NBA was complete in 30 min. At this time, 3-OH-ABA predominated over 3-ABA and could be isolated by precipitation. Overnight reaction gave complete conversion to 3-ABA. In contrast, reduction of 2-NBA was much slower at these conditions, so that overnight reaction was needed for complete conversion yielding mainly 2-OH-ABA. To accomplish further reduction to 2-ABA the reaction time had to be prolonged up to 2 days even when a more active catalyst was used. Acetylation of both 2- and 3-OH-ABA by pyruvonitrile in the presence of TBD-methyl polystyrene as a condensation reagent (Arlt et al., 2006) yielded 2-and 3-AcO-ABA, respectively. These esters are active intermediates generating nitrenium ions. Their reactions with NAC in aqueous solution gave low yields of corresponding mercapturic acids, which were separated from nonacidic products by pH directed extraction. The mercapturic acid derived from 3-NBA was identified by 1 H NMR, MS and UV spectra as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABAMA), that derived from 2-NBA was not obtained in sufficient amount for full structural characterisation. However, it showed the same major MS2 fragments as 3-ABA-MA with the only difference being the relative intensity of fragments at m/z 278 and 246. Moreover, according to calculated charge distribution of
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Fig. 1. LC–ESI-MS mass chromatograms, MS2 at m/z 246 for monitoring ABA: (A) blank urine; (B) urine of a rat dosed orally with 2 mg/kg of 2-NBA; (C) authentic 2-ABA; (D) urine of a rat dosed orally with 2 mg/kg of 3-NBA; (E) authentic 3-ABA.
benzanthronylnitrenium ions (Arlt et al., 2007), the most electrophilic carbon of the 2-benzanthronylnitrenium isomer is at the position 3. Therefore, the attack of the nucleophilic thiol group of NAC should proceed at this position. As a consequence, the structure of the mercapturic acid derived from 2-NBA can be tentatively assigned as 2-aminobenzanthron-3-ylmercapturic acid (2-ABAMA). Formation of 2- and 3-ABA-MA is shown in Scheme 1. 3.2. Identification of urinary metabolites Two expected urinary metabolites of each NBA isomer, namely, the corresponding aminobenzanthrone (ABA) and its N-acetyl derivative (AcABA), were detected in the extracts of rat urine by LC–ESI-MS and identified by comparison of their elution times and MS2 spectra with those of authentic samples. Thus, 2-NBA yielded 2-ABA and 2-AcABA, while 3-NBA analogically yielded 3-ABA and 3-AcABA. Mass chromatograms of the urine of rats dosed with 2and 3-NBA are shown in Figs. 1 and 2, respectively. No significant metabolite peaks were detected at m/z 261 for monooxygenated aminobenzanthrones. In the urine of rats dosed with 3-NBA a significant peak at m/z 407 was detected co-eluting with 3-ABA-MA. Its identity was confirmed by comparison of both positive and negative MS with that of authentic sample (Fig. 3). In contrast, no peak co-eluting with 2-ABA-MA was detected in the urine of rats dosed with 2-NBA (Fig. 4). 3.3. Excretion of aminobenzanthrones Only a minor proportion, less than 1%, of 2- and 3-NBA administered to rats was excreted in urine in the form of free ABA and/or their conjugates. The amounts of free 2- and 3-ABA as well as 2and 3-AcABA were determined in urine by LC–ESI-MS. In addition, total urinary 2- and 3-ABA (total = free plus released from their conjugates by acidic hydrolysis) were also determined by a general GC–MS method for analysis of urinary arylamines (Lind et al., 1997). Due to matrix effects observed in the GC–MS method, calibration by
Fig. 2. LC–ESI-MS mass chromatograms, MS2 at m/z 288 for monitoring of AcABA: (A) blank urine; (B) urine of a rat dosed orally with 2 mg/kg of 2-NBA; (C) authentic 2-AcABA; (D) urine of a rat dosed orally with 2 mg/kg of 3-NBA; (E) authentic 3AcABA.
internal or external standard was not satisfactory. Therefore, standard addition method was used for quantification. A typical mass chromatogram is shown in Fig. 5. Results are shown in Table 1. For both isomers the total ABA excretion as determined by GC–MS is in agreement with the sum of free ABA and AcABA determined by LC–ESI-MS. Urine was collected in two subsequent 24 h intervals after dosing. Compared to the first 24-h post-exposure period, only 5–10% of these amounts were excreted in the second 24-h post-exposure period. This indicates that low conversion to these expected metabolites observed during the first day cannot be explained by delayed metabolism or excretion. 3.4. Excretion of 3-ABA-MA Mercapturic acid identified in the urine of rats dosed with 3-NBA was quantified by LC–ESI-MS using authentic standard. Excretion of this metabolite amounted less than 1% of the dose. The main portion of it was excreted during the first 24 h post-exposure period, only a minor portion was found in urine collected between 24 and 48 h after dosing (Table 1). 4. Discussion The carcinogenic 3-NBA differs from its isomers including 2NBA in many ways. Quantum chemical calculations have shown that the nitrenium ion derived from 3-NBA is the most stable of all benzanthronylnitrenium ions. Moreover, significant positive correlation was found between nitrenium ion stabilities of four NBA isomers and their mutagenic activities in Salmonella (Reynisson et al., 2008). In general, nitrenium ion stability is considered to be an important factor in mutagenicity and carcinogenicity of the corresponding parent compound (nitroarene or arylamine) because long living reactive intermediates can more easily reach their targets in nuclear DNA (Borosky, 2007). However, the calculated difference of
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Table 1 Excretion of urinary metabolites in rats after a single oral dose (2 mg/kg) of 2-NBA or 3-NBA. Total 2- and 3-ABA, i.e., sum of free plus conjugated forms were determined by GC–MS, other analyses were performed by LC–MS. Metabolite
Compound administered 2-NBA 2-ABA total
3-NBA 2-ABA free
Urinary excretion [% of dose; mean ± SE, n = 3] 0.32 ± 0.06 0.12 ± 0.05 0–24 h 24–48 h 0.008 ± 0.004 <0.01a a
2-AcABA
3-ABA total
3-ABA free
3-AcABA
3-ABA-MA
0.24 ± 0.11 <0.03a
0.83 ± 0.40 0.04 ± 0.01
0.10 ± 0.06 <0.01a
0.41 ± 0.25 <0.02a
0.49 ± 0.15 0.02 ± 0.01
Below the limit of quantification of the LC–ESI-MS method used, which ranged from 1.5 to 4 ng/mL for each analyte.
ca. 9 kcal/mol in the nitrenium ion energies of 2- and 3-NBA is very unlikely to account for the whole difference in their mutagenic and carcinogenic potentials. Our experiments on palladium catalysed reduction of 2- and 3NBA showed a markedly different reactivity, 3-NBA being much more reactive. In contrast, metabolic yield of total 2-ABA was only slightly lower than that of 3-ABA. In vivo, reductive metabolic activation of 3-NBA is catalysed mainly by NQO1 (Arlt et al., 2005; Stiborová et al., 2006). Despite strong binding of both 2- and 3-NBA to this enzyme, only 3-NBA seems to be efficiently metabolically activated to the corresponding nitrenium ion. This was demonstrated by strikingly different extent of DNA adduct formation both in vitro and in vivo (Arlt et al., 2007). Convincing explanation of this paradox was provided by Stiborová et al. Molecular docking of the two isomers into the active site of NQO1 showed similar
binding affinities but, unlike for 3-NBA, orientation of the 2-NBA molecule in the enzyme-substrate complex does not allow reduction of its nitro group, so that metabolic activation of 2-NBA by this enzyme cannot occur (Stiborová et al., 2010). This seems to be the key difference between metabolism of 2-NBA and 3-NBA, responsible for their strikingly different biological activities as well as for the lack of formation of 2-ABA-MA by biotransformation of 2-NBA. It is most likely that the reduction of 2-NBA to 2-ABA is catalysed by other enzyme(s) than NQO1, and the ultimate electrophilic intermediate, 2-benzantronylnitrenium ion, which is less stable than 3-benzanthronylnitrenium ion (Reynisson et al., 2008), is formed in a much smaller extent during this biotransformation. This explanation is supported also by in vitro DNA binding studies. In various human cells binding of 2-NBA was up to 14-fold lower than that of 3-NBA, as determined by 32 P-postlabelling (Arlt et al., 2007). In the present study we observed a marked difference between 2- and 3-NBA in the mercapturic acid formation, with only the carcinogenic 3-NBA being demonstrated as a mercapturic acid precursor. Mercapturic acids are formed by glutathione conjugation of electrophilic species, very often reactive metabolites of various xenobiotics. Therefore, formation of these detoxication products is
Fig. 3. LC–ESI-MS mass chromatograms, MS2 at m/z 407 for monitoring of 3-ABAMA: (A) blank urine; (B) urine of a rat dosed with 2 mg/kg of 3-NBA; (C) blank urine spiked with authentic 3-ABA-MA. MS2 of 3-ABA-MA is shown in the insert.
Fig. 4. LC–ESI-MS mass chromatograms, MS2 at m/z 407 for monitoring of 2-ABAMA: (A) blank urine; (B) urine of a rat dosed with 2 mg/kg of 2-NBA; (C) blank urine spiked with authentic 2-ABA-MA. MS2 of 2-ABA-MA is shown in the insert.
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5. Conclusion The observed difference between 2- and 3-NBA in the mercapturic acid formation in rats in vivo reflects differences in the reductive bioactivation pathway, which may be critical for their mutagenic and carcinogenic potential. If confirmed also in humans, 3-ABA-MA would be a promising biomarker of exposure to and effective dose of 3-NBA. Conflict of interest None. Acknowledgements Financial support of this work by Grant Nos. 2B08051 and MSM 604 613 73 01 from the Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged. We are also grateful to Mr. Jiˇrí Kosina for the HRMS measurement. References
Fig. 5. GC–MS determination of 3-ABA in the acid hydrolyzed rat urine: (A) control urine; (B) urine of a rat dosed orally with 3-NBA at 2 mg/kg; (C) urine of a rat dosed with 3-NBA, spiked with 3-ABA, 0.1 g/mL. 3-ABA was measured as a HFB derivative at m/z 441.
closely related to toxicity of the corresponding xenobiotic, particularly to its mutagenic and carcinogenic potential. Thus, formation of 3-ABA-MA seems to reflect strong mutagenic and carcinogenic potential of 3-NBA, which is in fact much stronger than that of 2-NBA. Regardless of the expected common formation of mercapturic acids derived from mutagenic nitroarenes or arylamines, these in fact appear to be either exceptional or mostly unexplored. To our knowledge, since the early studies of Boyland et al. (1962, 1963) mercapturic acid formation from any arylnitrenium ion has not been reported. Thus, our finding of 3-ABA-MA seems to be the first report of an arylnitrenium derived mercapturic acid formation in vivo after 48 years. Taking into account the toxicological relevance of mercapturic acid pathway our observation indicates that there is a need for further investigation of possible mercapturic acid formation from other mutagenic and/or carcinogenic nitroaromatics and aromatic amines. In the case of 3-NBA, the identified mercapturic acid seems to be a promising specific biomarker of exposure and a valuable surrogate biomarker of effective dose. It remains to be established whether or not it is formed also in humans. Urinary metabolites detected in this study account for only a minor portion of the dose. No other significant metabolites have been detected by the methods used. Further studies are needed to describe the overall metabolic fate of 2- and 3-NBA. Such studies would be greatly facilitated by the administration of radiolabelled substrates.
Angerer, J., Ewers, U., Wilhelm, M., 2007. Human biomonitoring: state of the art. Int. J. Hyg. Environ. Health 210, 201–228. Arlt, V.M., 2005. 3-Nitrobenzanthrone, a potential human cancer hazard in diesel exhaust and urban air pollution: review of the evidence. Mutagenesis 20, 399–410. Arlt, V.M., Bieler, C.A., Mier, W., Wiessler, M., Schemiser, H.H., 2001. DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by 32 P-postlabeling. Int. J. Cancer 93, 450–454. Arlt, V.M., Stiborová, M., Henderson, C.J., Osborne, M.R., Bieler, C.A., Frei, E., Martínek, V., Sopko, B., Wolf, C.R., Schmeiser, H.H., Phillips, D.H., 2005. The environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosol. Cancer Res. 65, 2644–2652. Arlt, V.M., Sorg, B.L., Osborne, M., Kawanishi, M., Kanno, T., Yagi, T., Phillips, D.H., Takamura-Enya, T., 2006. Identification of three major DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in rat lung at the C8 and N2 position of guanine and N6 position of adenine. Int. J. Cancer 118, 2139–2146. Arlt, V.M., Glatt, H., da Costa, G.G., Reynisson, J., Takamura-Enya, T., Phillips, D.H., 2007. Mutagenicity and DNA adduct formation by the urban air pollutant 2nitrobenzanthrone. Toxicol. Sci. 98, 445–457. Bieler, C.A., Cornelius, M., Klein, R., Arlt, V.M., Wiessler, M., Phillips, D.H., Schmeiser, H.H., 2005. DNA adduct formation by the environmental contaminant 3nitrobenzanthrone after intratracheal instillation in rats. Int. J. Cancer 118, 833–838. Bieler, C.A., Cornelius, M., Stiborová, M., Arlt, V.M., Wiessler, M., Phillips, D.H., Schmeiser, H.H., 2007. Formation and persistence of DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in target and non-target organs after intratracheal instillation in rats. Carcinogenesis 28, 1117–1121. Borlak, J., Hansen, T., Yuan, Z.-X., Sikka, H.C., Kumar, S., Schmidbauer, S., Frank, H., Jacob, J., Seidel, A., 2000. Metabolism and DNA-binding of 3-nitrobenzanthrone in primary rat alveolar type II cells, in human fetal bronchila, rat epithelial and mesenchymal cell lines. Polycycl. Aromat. Compd. 21, 73–86. Borosky, G.L., 2007. Ultimate carcinogenic metabolites from aromatic and heteroaromatic amines: a computational study in relation to their mutagenic potency. Chem. Res. Toxicol. 20, 171–180. Boyland, E., Manson, D., Nery, R., 1962. The reaction of phenylhydroxylamine and 2-napthylhydroxylamine with thiols. J. Chem. Soc. 606, 611. Boyland, E., Manson, D., Nery, R., 1963. Mercapturic acids as metabolites of aniline and 2-naphthylamine. Biochem. J. 86, 263–271. da Costa, G.G., Singh, R., Arlt, V.M., Mirza, A., Richards, M., TakamuraEnya, T., Schmeiser, H.H., Farmer, P.B., Phillips, D.H., 2009. Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC–electrospray tandem mass spectrometry. Chem. Res. Toxicol. 22, 1860–1868. Enya, T., Suzuki, H., Watanabe, T., Hirayama, T., Hisamatsu, Y., 1997. 3Nitrobenzanthrone, a powerful bacterial mutagen and suspected human carcinogen found in diesel exhausts and airborne particulates. Environ. Sci. Technol. 31, 2772–2776. Enya, T., Kawanishi, M., Suzuki, H., Matsui, S., Hisamatsu, Y., 1998. An unusual DNA adduct derived from the powerfully mutagenic environmental contaminant 3nitrobenzanthrone. Chem. Res. Toxicol. 11, 1460–1467. Hansen, T., Seidel, A., Borlak, J., 2007. The environmental carcinogen 3nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells. Toxicol. Appl. Pharmacol. 221, 222–234.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Carcinogenic 3-nitrobenzanthrone but not 2-nitrobenzanthrone is metabolised to an unusual mercapturic acid in rats a ˇ Igor Linhart a,∗ , Jaroslav Mráz b , Iveta Hanzlíková b , Alexandra Silhánková , Emil Frantík b , Michal Himl a a b
Department of Organic Chemistry, Faculty of Chemical Technology, Institute of Chemical Technology, Prague, Technická 1905, CZ-166 28 Prague, Czech Republic National Institute of Public Health, Sˇ robárova 48, CZ-100 42 Prague, Czech Republic
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 18 November 2011 Accepted 19 November 2011 Available online 26 November 2011 Keywords: Nitrobenzanthrones Environmental carcinogens Biotransformation Urinary metabolites Mercapturic acid
a b s t r a c t 3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspect human carcinogen found in diesel exhaust. Its isomer 2-nitrobenzanthrone (2-NBA) has also been found in ambient air. These isomers differ in mutagenicity in Salmonella by 2–3 orders of magnitude. To identify their urinary metabolites and also to assess the assumed differences in their excretion, rats were dosed orally with 2 mg/kg b.w. of either 2-NBA or 3-NBA. Their urine was collected for two consecutive days after dosage. Both LC–ESI-MS and GC–MS confirmed formation of the corresponding aminobenzanthrones (ABA). Excretion of these metabolites within the first day after dosing with 2- and 3-ABA amounted to 0.32 ± 0.06 and 0.83 ± 0.40% of the doses, respectively, while the excretion within the second day was by one order of magnitude lower. A novel mercapturic acid metabolite of 3-NBA was identified in urine by LC–ESI-MS as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABA-MA) by comparison with the authentic standard. Its excretion amounted to 0.49 ± 0.15 and 0.02 ± 0.01% of dose within the first and second day after dosing, respectively. In contrast, no mercapturic acid was detected in the urine of rats dosed with 2-NBA. Observed difference in the mercapturic acid formation between 2and 3-NBA is a new distinctive feature reflecting differences in the critical step of their metabolism, i.e., benzanthronylnitrenium ion formation that is intrinsically associated with biological activities of these two isomers. Moreover, 3-ABA-MA is a promising candidate biomarker of exposure to the carcinogenic 3-NBA. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Both 2- and 3-nitro-7H-benz[de]anthracen-7-one (2- and 3nitrobenzanthrone, NBA), like numerous other polycyclic nitroaromatics, are important environmental pollutants emitted from diesel engines. They are found mainly adsorbed on the surface of airborne particulate matter and are of great concern due to their mutagenic and carcinogenic potential (Enya et al., 1997; Arlt, 2005; Arlt et al., 2007). Although both are mutagenic in Salmonella typhimurium striking differences exist in their mutagenic potential with 3-NBA being by 2–3 orders of magnitude more mutagenic than the 2-nitro isomer (Takamura-Enya et al., 2006). Number of revertants induced in the Ames test by 3-NBA is comparable to that of 1,8-dinitropyrene, one of the most potent mutagens known (Purohit and Basu, 2000; Arlt, 2005). Carcinogenicity of 3-NBA was demonstrated in an experiment on rats following intratracheal instillation (Nagy et al., 2005). Numerous 32 P-postlabelling and MS studies have shown formation of DNA adducts in rats dosed with
∗ Corresponding author. Tel.: +420 220 444 165; fax: +420 220 444 288. E-mail address: [email protected] (I. Linhart). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.11.017
3-NBA as well as in experiments with metabolically activated 3NBA in vitro (Bieler et al., 2005, 2007; Arlt et al., 2001, 2006; da Costa et al., 2009). DNA adducts were formed also from 2-NBA in rat lungs in vivo, although only at very high doses of 2-NBA (Arlt et al., 2007). 3-NBA is metabolically reduced to 3-(hydroxyamino) benzanthrone (3-OH-ABA), which is either activated by Oacetylation and sulphate conjugation or further reduced to 3-aminobenzanthrone (3-ABA). 3-ABA was identified in the urine of mining workers heavily exposed to diesel emissions (Seidel et al., 2002). Both acetate and sulphate conjugates dissociate to electrophilic 3-benzanthronylnitrenium ion, which is the ultimate DNA reactive species (Scheme 1). Reduction of 3-NBA is catalysed mainly by NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) (see Scheme 2). In general, electrophilic species are detoxified by mercapturic acid pathway (MAP) initiated by conjugation with glutathione (GSH), followed by subsequent cleavage of glutamine and glycine, and terminated by acetylation to yield S-substituted N-acetylcysteines, mercapturic acids, which are excreted in urine. Mercapturic acids can serve as valuable biomarkers of exposure reflecting electrophilic reactivity of xenobiotics and/or
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Scheme 1. Preparation of the mercapturic acids derived from 2- and 3-NBA by reaction of the corresponding activated OH-ABAs with NAC.
their metabolites (for reviews see Haufroid and Lison, 2005; Angerer et al., 2007). Numerous in vitro studies on the metabolic activation of 3-NBA were published (Borlak et al., 2000; Arlt, 2005; Hansen et al., 2007; Stiborová et al., 2006), however, only one urinary metabolite, 3aminobenzanthrone, has been identified as yet (Seidel et al., 2002). Arylation of N-acetylcysteine (NAC) and GSH by activated hydroxyaminoarenes (Boyland et al., 1962; Manson, 1972; Ketterer et al., 1979) as well as by benzidine upon oxidative activation by horseradish peroxidase (Josephy and Iwaniw, 1985) has been described long ago. Therefore, it is rather surprising that, to our knowledge, very few mercapturic acids formed via arylnitrenium ions in vivo have been reported so far, namely, 2and 4-aminophenylmercapturic acids derived from aniline and 2amino-1-naphthylmercapturic acid derived from 2-naphthylamine (Boyland et al., 1963). The aim of the present work was to identify urinary metabolites of both 2- and 3-NBA, in particular those produced through the formation of nitrenium ion intermediates. The amounts of mercapturic acids possibly found after administration of 2- and 3-NBA would reflect the extent of formation of the corresponding benzanthronylnitrenium ions and, indirectly, the extent of toxic insult caused by each NBA isomer.
2. Materials and methods 2.1. Materials Acetonitrile for LC/MS Chromasolv was from Merck, formic acid, puriss. p.a. from Fluka. Re-distilled water was used for LC/MS and solid phase extraction. Tetrahydrofuran (THF) was dried by distillation from sodium and benzophenone. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene bound to polystyrene (TBD-methyl polystyrene) was from Novabiochem, Germany. Heptafluorobutyric anhydride (HFBA) was from Fluka. Other chemicals were of synthetic or analytical grade and were used as received.
2.2. Syntheses of nitrobenzanthrones and their expected metabolites 2-Nitrobenzanthrone (2-NBA) was prepared by a published multi-step synthesis (Suzuki et al., 1997) with a modification in the key step. Instead of Ullmann reaction, which gave low yields of the key intermediate 1-(2methoxycarbonylphenyl)-3-nitronaphthalene, Suzuki coupling (Miyaura et al., 1979) of 1-iodo-3-nitronaphthalene with 2-(methoxycarbonyl)phenylboronic acid was used.
3-Nitrobenzanthrone (3-NBA) was obtained by direct nitration of benzanthrone with fuming nitric acid as described in the literature (Enya et al., 1998). 2-Aminobenzanthrone (2-ABA). Palladium catalyst (10 mg of 10% Pd on charcoal, Degussa type containing ca. 50% of water) was activated by heating at 90 ◦ C for 1 h in a vacuum. Argon was then introduced into the reaction flask and 2-NBA (15 mg, 54.5 mol), diglym (30 mL) and hydrazine hydrate (75 L, 77 g, 1.54 mol) were added. The reaction mixture was stirred at room temperature under argon and the course of reaction was monitored by HPLC with a PDA detector. After 2 days conversion of 2-NBA was complete. The catalyst was then filtered off and the filtrate was evaporated in a vacuum to dryness. Crystallisation of the residue from ethanol yielded 9.5 mg (71%) of orange crystals identified by comparing their 1 H NMR spectrum with that reported in the literature as 2-ABA (Takekawa et al., 2002). ESI-MS: m/z 246 (M+H)+ , 268 (M+Na)+ . MS2 : m/z 246 → 228 (MH−H2 O)+ , 229 (MH−NH3 )+ ; 230 (MH−O)+ ; 217 (MH−COH)+ ; 218 (MH−CO)+ , 219 (MH−C2 H2 )+ , 201 (MH−CONH3 )+ . UV: max = 442, 375, 303 and 230 nm. 2-Acetamidobenzanthrone (N-acetyl-2-aminobenzanthrone, 2-AcABA). 2-ABA (10 mg, 41 mol) was refluxed in acetic anhydride (1.5 mL) for 4 h. Reaction mixture was then evaporated to dryness in a vacuum. Re-crystallisation of the residue from aqueous ethanol yielded 6.5 mg (56%) of greenish-yellow crystals. 1 H NMR spectrum (CDCl3 ): ı = 2.40 (s, 3H, CH3 CO); 7.61 (td, J = 7.5 and 1.0 Hz, 1H, C9-H); 7.76 (td, J = 7.7 and 1.5 Hz, 1H, C10-H); 7.82 (d, J = 1.8 Hz, 1H, C3-H); 7.84 (dd, J = 8.2 and 7.6 Hz, 1H, C5-H); 8.19 (d, J = 1.9 Hz, 1H, C1-H); 8.22 (dd, J = 8.2 and 1.2 Hz, 1H, C11-H); 8.28 (d, J = 8.2 Hz, 1H, C4-H); 8.52 (dd, J = 7.9 and 1.1 Hz, 1H, C8-H); 8.80 (dd, J = 7.3 and 1.4 Hz, 1H, C6-H). ESI-MS: m/z 288 (M+H)+ , MS2 : m/z 288 → 246 (MH−CH2 CO)+ . UV: max = 389, 307, 256 and 240 nm. 2-(Hydroxyamino)benzanthrone (2-OH-ABA). To a suspension of 2-NBA (15 mg, 54.5 mol) in diglym (30 mL) palladium catalyst (15 mg of 5% Pd on charcoal) and hydrazine hydrate (75 L, 77 g, 1.54 mol) was added. The reaction mixture was then stirred overnight under argon at the room temperature. Conversion of 2-NBA amounted to more than 90% as determined by HPLC with PDA detector. After filtering off the catalyst, the solvent was evaporated in a vacuum to dryness, and re-suspended in 20 mL of a 1:1 ethyl acetate–chloroform mixture. The solid was filtered off to yield 5 mg (35%) of a greenish yellow powder identified as 2-OH-ABA. 1 H NMR spectrum (DMSO-d6 ): ı = 7.51 (bs, 1H, NH); 7.64 (t, J = 7.5 Hz, 1H, C9-H); 7.75 (t, J = 7.8 Hz, 1H, C10-H); 7.86 (t, J = 7 Hz, 1H, C5-H); 8.22 (d, J = 2 Hz, 1H, C3-H); 8.25 (d, J = 7.5 Hz, 1H, C4-H); 8.32 and 8.35 (d, J = 6.7 Hz, 2H, C6-H and C8-H); 8.41 (d, J = 8.2 Hz, 1H, C11-H); 8.78 (d, J = 1.8 Hz, 1H, C1-H); 8.88 (bs, 1H, OH). ESI-MS: m/z 262 (M+H)+ ; MS2 : 262 → 245 (MH−NH3 )+ , 244 (MH−H2 O)+ , 234 (MH−CO)+ , 217 (MH−CO−NH3 )+ . UV: max = 430, 375, 297 and 224 nm. 3-Aminobenzanthrone (3-ABA). 3-NBA (20 mg, 72.7 mol) was dissolved in 20 mL of diglym, palladium catalyst (20 mg of 5% Pd on charcoal) and hydrazine hydrate (80 L, 82 mg, 1.65 mol) was added and the reaction mixture was stirred overnight under argon at room temperature. The catalyst was then filtered off, the filtrate was evaporated in a vacuum to dryness and the residue re-dissolved in 5 mL of ethanol. Red precipitate formed after dilution of this solution with water was filtered off and dried over P4 O10 . Dark red powder of 3-ABA obtained (9 mg, 51%) showed 1 H NMR spectrum, which was in agreement with published data (Safronov and Traven, 1993).
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Scheme 2. Biotransformation of 2- and 3-NBA to the urinary metabolites found in rats in vivo. NQO1 – NAD(P)H:quinone oxidoreductase 1, NAT – N-acetyltransferase, SULT – sulfotransferase; MAP – mercapturic acid pathway.
ESI-MS: m/z 246 (M+H)+ , 268 (M+Na)+ . MS2 : m/z 246 → 228 (MH−H2 O)+ , 229 (MH−NH3 )+ ; 230 (MH−O)+ ; 217 (MH−COH)+ ; 218 (MH−CO)+ , 219 (MH−C2 H2 )+ , 201 (MH−CONH3 )+ . ESI-MS: m/z 246 (M+H)+ ; 268 (M+Na)+ . UV: max = 507, 280 and 218 nm. 3-Acetamidobenzanthrone (N-acetyl-3-aminobenzanthrone, 3-AcABA) was obtained as described for 2-AcABA as yellow-brown crystals from aqueous ethanol. 1 H NMR spectrum (CDCl3 ): ı = 2.37 (s, 3H, CH3 CO); 7.56 (d, J = 7.9 Hz, 1H, C2-H); 7.6 (t, J = 8 Hz, 1H, C9-H); 7.78 (td, J = 7.8 and 1.5 Hz, 1H, C5-H); 7.85 (t, J = 8 Hz, 1H, C10-H); 8.07 (dd, J = 8 and 1.2 Hz, 1H, C11-H); 8.34 (d, J = 8.2 Hz, 1H, C1-H); 8.50 (dd, J = 8 and 1.2 Hz, 1H, C8-H); 8.52 (d, J = 7.8 Hz, 1H, C6-H); 8.80 (dd, J = 7.5 and 1.2 Hz, 1H, C4-H). ESI-MS: m/z 288 (M+H)+ , MS2 : m/z 288 → 246 (MH−CH2 CO)+ . UV: max = 416, 310, 262 and 237 nm.
3-(Hydroxyamino)benzanthrone (3-OH-ABA) was prepared by reduction of 3-NBA with hydrogen on palladium as described earlier (Osborne et al., 2005). (3-aminobenzanthron-2N-Acetyl-S-(3-aminobenzanthron-2-yl)cysteine ylmercapturic acid, 3-ABA-MA). This mercapturic acid was obtained by the reaction of 3-(acetoxyamino)-benzanthrone (3-AcO-ABA) with NAC. 3-AcO-ABA was prepared by reacting 25 mg (96 mol) of 3-OH-ABA with pyruvonitrile in presence of TBD-methyl polystyrene as published previously (Arlt et al., 2006). Then, its solution in 20 mL of TFH was added immediately to 5 mL of aqueous solution of NAC (19 mg, 117 mol) pH of which was adjusted to 7.6. The reaction mixture was left standing overnight at the room temperature, thereafter diluted with chloroform (30 mL) and extracted with 20 mL of 0.1 M Na2 HPO4 (pH 9.1). Aqueous solution was acidified to pH 3.2 with 1 M HCl and extracted twice with 10 mL portions of ethyl acetate. Evaporation of the ethyl acetate extract to dryness in a vacuum yielded 2 mg of crude product. Combined crude products from two
I. Linhart et al. / Toxicology Letters 208 (2012) 246–253 experiments were purified by semi-preparative HPLC on a 250 × 10 mm I.D. column Phenomenex Luna C18(2), 10 m particle size. The column was eluted at a flow rate of 2.8 mL/min with a gradient of acetonitrile in aqueous formic acid (0.15%). Concentration of acetonitrile increased linearly from 35.5% to 50% within 20 min. The peak of 3-ABA-MA was collected at 17.8 min. 1 H NMR (acetone-d6 ): ı = 1.90 (s, 3H, CH3 CO); 3.25 (dd, J = 7.6 and 14 Hz, 1H, CH2 S); 3.41 (dd, J = 4.1 and 14 Hz, 1H, CH2 S); 4.75 (m, 1H, CHN); 6.92 (bs, 2H, NH2 ); 7.42 (t, J = 7.6 Hz, 1H, C10-H); 7.76 (t, J = 7.8 Hz, 1H, C5-H); 7.83 (t, J = 7.2, 1H, C9-H); 8.37 (dd, J = 1.5 and 7.6 Hz, 1H, C11-H); 8.51 (d, J = 7.9 Hz, 1H, C8-H); 8.74 (d, J = 7.3 Hz, 1H, C4-H or C6-H); 8.76 (d, J = 8 Hz, 1H, C4-H or C6-H); 8.78 (s, 1H, C1-H); 7.57 (bd, J = 7.5 Hz, 1H, NH). ESI-MS: m/z 407 (M+H)+ , 405 (M−H)− ; MS2 : 407 → 365 (MH−CH2 CO)+ , 278 (MH−CH2 C(NHCOCH3 )COOH)+ , 246 (MH−SCH2 C(NHCOCH3 )COOH)+ , 405 → 276 (M−SCH2 CH(NHCOCH3 )COO)− ; HRMS: calculated for C22 H19 N2 O4 S+ 407.10600; found 407.10587. UV: max = 504, 288 and 226 nm. Reaction of 2-(acetoxyamino)benzanthrone (2-AcO-ABA) with N-acetylcysteine. THF solution (12 mL) of 2-AcO-ABA that was prepared from 15 mg (57 mol) of 2-OH-ABA as described above for 3-AcO-ABA, was immediately added to a solution of 11 mg (66 mol) of NAC in 3 mL of water (pH 7.6). The mercapturic acid formed was isolated as described above for 3-ABA-MA. Its amount was not sufficient for full structure characterisation, however, fragmentation in ESI-MS2 of both (M+H)+ and (M−H)− afforded the same ions as 3-ABA-MA, thus suggesting strongly that the product is 2-ABA-MA. ESI-MS: m/z 407 (M+H)+ , 405 (M−H)− ; MS2 : 407 → 365 (MH−CH2 CO)+ , 278 (MH−CH2 C(NHCOCH3 )COOH)+ , 246 (MH−SCH2 C(NHCOCH3 )COOH)+ , 405 → 276 (M−SCH2 CH(NHCOCH3 )COO)− . UV: max = 451, 377, 314, 235 nm.
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another 5 min. The flow rate was 200 L/min. Mass spectra (positive ions) were taken in the range of m/z 150–450. MS2 spectra were taken at m/z 246 for 2- and 3-ABA with a collision energy of 36 V and at m/z 288 for 2- and 3-Ac-ABA with a collision energy of 25 V. Transitions at m/z 246 → 219, 246 → 218 and 246 → 217 were used for quantification of 2-and 3-ABA, transition at m/z 288 → 246 for quantification of 2- and 3-ABA. Calibration samples were prepared by spiking blank urine to provide analyte concentrations from 1.5 to 30 ng/mL for 2- and 3-ABA and from 3 to 30 ng/mL for 2- and 3-AcABA. The calibration curves were linear (R2 > 0.99). 2.5.2. Acidic metabolites Urine samples were neutralised to pH 7.0–7.5 with 0.1 M HCl and filtered through 0.45 m Nylon membrane filters. Filtrates (1 mL aliquots) were diluted with 2 mL of water and poured onto pre-conditioned 60 mg Oasis HLB SPE columns (Waters GmbH). The columns were washed with 3 mL of water followed by 3 mL of 20% aqueous methanol and finally eluted with 3 mL of methanol. The eluates were evaporated to dryness at 40 ◦ C in the stream of nitrogen, residues were re-dissolved in 300 L of 40% acetonitrile for LC–ESI-MS analysis. Aliquots (10 L) were injected onto a BDS Hypersil C18 column (150 × 2 mm I.D., 5 m particle size; Thermo). The column was eluted with a gradient of acetonitrile in 0.075% aqueous formic acid. Concentration of acetonitrile was increased linearly from 9% to 65% within 20 min and than to 83% within another 2 min. Mass spectra (positive ions) were taken in the range of m/z 150–500. MS2 spectra were taken at m/z 407 for ABA-MA with a collision energy of 32 V. Additionally, negative ion spectra at m/z 405 with a collision energy of 21 V were taken in a separate run. Transition at m/z 407 → 278 in the positive ion mode was used for quantification of 3-ABA-MA. Calibration samples were prepared by spiking blank urine to provide analyte concentrations from 5.2 to 208 ng/mL. The calibration curve was linear in this range of concentrations (R2 > 0.99).
2.3. Analytical instrumentation LC/MS analyses were carried out on a Thermo Scientific LXQ linear trap mass spectrometer in tandem with a Janeiro LC system consisting of two Rheos 2200 pumps and CTC PAL autosampler. ESI in both positive and negative ion modes was used. Ion source capillary temperature was set to 300 ◦ C. In some experiments a Thermo Scientific Surveyor PDA detector was used instead of or in series with the MS detector. NMR spectra were taken on a Varian Gemini 300 MHz spectrometer. High resolution MS was measured on a Thermo Scientific LTQ Orbitrap Velos spectrometer. GC/MS analyses were performed on a 7890 gas chromatograph equipped with 5975C inert XL EI/CI MSD (Agilent). Samples were introduced onto a capillary column DB-5ms, 30 m × 0.25 mm I.D. with 0.25 m film thickness (Agilent). Injector was set in a splitless injection mode. The column oven temperature was held at 80 ◦ C for 1 min, then raised at 30 ◦ C/min to 320 ◦ C and held at this temperature for 5 min. Injector temperature was 250 ◦ C. Helium was used as the carrier gas at a linear velocity of 30 cm/s. MS detector was operated in the electron ionisation (EI) mode at 70 eV, and was set in a SIM mode at m/z 441 for determination of HFB derivatives of ABA. 2.4. Animal treatment Adult male Wistar rats, average weight 276 ± 6 g (mean ± SD, n = 8) were placed individually into glass metabolic cages with free access to pelleted food and water. To enhance diuresis, sucrose (8 mg/mL) was added to the drinking water. Control urine was collected for 24 h. 2-NBA and 3-NBA were dissolved in DMSO at concentrations of 0.5 and 1 mg/mL, respectively. Concentration of these solutions was limited by solubility. Three rats were dosed by gavage with 4 mL/kg of the 2-NBA solution, another three rats were dosed with 2 mL/kg of the 3-NBA solution, so that both groups received a dose of 2 mg/kg of one NBA isomer. Two control animals were dosed with DMSO only. Urine was collected in the time intervals from 0 to 24 h and from 24 to 48 h after administration. During sample collection the urine was filtered through a gauze filter to remove pieces of faeces and crumbs of food pellets. The walls of the metabolic cages were rinsed with distilled water and resulting solution was added to the main portion of collected urine. Samples were stored at −20 ◦ C until analysed. Animal experiments were approved by the Commission for Animal Protection of the NIPH and of the Czech Ministry of Health. 2.5. Analyses of urine by LC–ESI-MS 2.5.1. Non-acidic metabolites Urine samples were neutralised to pH 7.5–7.8 with 0.1 M HCl and filtered through 0.2 m Nylon membrane filters. Filtrates (1 mL aliquots) were diluted with 2 mL of water and poured onto 60 mg Oasis HLB SPE columns (Waters GmbH), which were pre-conditioned by washing them with 3 mL of methanol followed by 3 mL of water. The columns were washed subsequently with 3 mL of 10% aqueous methanol and 3 mL of 50% methanol and eluted with 3 mL of 1:1 mixture of methanol and acetonitrile. The eluates were evaporated to dryness at 40 ◦ C in the stream of nitrogen and the residues were re-dissolved in 300 L of 40% acetonitrile for LC–ESI-MS analysis. Aliquots (10 L) of the urine extracts were injected onto a BDS Hypersil C18 column (150 × 2.1 mm I.D., 5 m particle size). The column was eluted with a gradient of acetonitrile in 0.075% aqueous formic acid. Concentration of acetonitrile increased linearly from 9% to 83% within 20 min and was then kept constant for
2.6. Determination of total 2- and 3-ABA in urine by GC–MS Urine was analysed for total (free plus conjugated) 2- or 3-ABA by a modified method as described in Lind et al. (1997). Urine samples (1 mL) were acidified with 1 mL of 3 M sulphuric acid the mixture was then vortexed in a 10 mL screw-cup tube and heated at 100 ◦ C for 2 h. After cooling, 10 M NaOH (2 mL) was added and the alkalinised sample was extracted with toluene (2 mL). The organic layer was separated, HFBA (20 L) was added and resulting mixture was heated at 50 ◦ C for 1 h. The organic layer was washed with 0.1 M phosphate buffer pH 7 (1.5 mL) and then taken in a vacuum concentrator to dryness. The residue was taken up in toluene (100 L) and analysed by GC/MS. Quantification of ABA in urine was carried out using standard addition method. Thus, with each sample, another aliquot was worked-up in parallel, to which aqueous solution of the respective ABA (1 g/mL, 100 L) was added.
3. Results 3.1. Preparation of authentic standards Reduction of 2- and 3-NBA by hydrogen on palladium yielded corresponding OH-ABA and, when the reaction time was prolonged, ABA as the end-product. The two NBA isomers significantly differed in their reactivity. Conversion of 3-NBA was complete in 30 min. At this time, 3-OH-ABA predominated over 3-ABA and could be isolated by precipitation. Overnight reaction gave complete conversion to 3-ABA. In contrast, reduction of 2-NBA was much slower at these conditions, so that overnight reaction was needed for complete conversion yielding mainly 2-OH-ABA. To accomplish further reduction to 2-ABA the reaction time had to be prolonged up to 2 days even when a more active catalyst was used. Acetylation of both 2- and 3-OH-ABA by pyruvonitrile in the presence of TBD-methyl polystyrene as a condensation reagent (Arlt et al., 2006) yielded 2-and 3-AcO-ABA, respectively. These esters are active intermediates generating nitrenium ions. Their reactions with NAC in aqueous solution gave low yields of corresponding mercapturic acids, which were separated from nonacidic products by pH directed extraction. The mercapturic acid derived from 3-NBA was identified by 1 H NMR, MS and UV spectra as N-acetyl-S-(3-aminobenzanthron-2-yl)cysteine (3-ABAMA), that derived from 2-NBA was not obtained in sufficient amount for full structural characterisation. However, it showed the same major MS2 fragments as 3-ABA-MA with the only difference being the relative intensity of fragments at m/z 278 and 246. Moreover, according to calculated charge distribution of
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Fig. 1. LC–ESI-MS mass chromatograms, MS2 at m/z 246 for monitoring ABA: (A) blank urine; (B) urine of a rat dosed orally with 2 mg/kg of 2-NBA; (C) authentic 2-ABA; (D) urine of a rat dosed orally with 2 mg/kg of 3-NBA; (E) authentic 3-ABA.
benzanthronylnitrenium ions (Arlt et al., 2007), the most electrophilic carbon of the 2-benzanthronylnitrenium isomer is at the position 3. Therefore, the attack of the nucleophilic thiol group of NAC should proceed at this position. As a consequence, the structure of the mercapturic acid derived from 2-NBA can be tentatively assigned as 2-aminobenzanthron-3-ylmercapturic acid (2-ABAMA). Formation of 2- and 3-ABA-MA is shown in Scheme 1. 3.2. Identification of urinary metabolites Two expected urinary metabolites of each NBA isomer, namely, the corresponding aminobenzanthrone (ABA) and its N-acetyl derivative (AcABA), were detected in the extracts of rat urine by LC–ESI-MS and identified by comparison of their elution times and MS2 spectra with those of authentic samples. Thus, 2-NBA yielded 2-ABA and 2-AcABA, while 3-NBA analogically yielded 3-ABA and 3-AcABA. Mass chromatograms of the urine of rats dosed with 2and 3-NBA are shown in Figs. 1 and 2, respectively. No significant metabolite peaks were detected at m/z 261 for monooxygenated aminobenzanthrones. In the urine of rats dosed with 3-NBA a significant peak at m/z 407 was detected co-eluting with 3-ABA-MA. Its identity was confirmed by comparison of both positive and negative MS with that of authentic sample (Fig. 3). In contrast, no peak co-eluting with 2-ABA-MA was detected in the urine of rats dosed with 2-NBA (Fig. 4). 3.3. Excretion of aminobenzanthrones Only a minor proportion, less than 1%, of 2- and 3-NBA administered to rats was excreted in urine in the form of free ABA and/or their conjugates. The amounts of free 2- and 3-ABA as well as 2and 3-AcABA were determined in urine by LC–ESI-MS. In addition, total urinary 2- and 3-ABA (total = free plus released from their conjugates by acidic hydrolysis) were also determined by a general GC–MS method for analysis of urinary arylamines (Lind et al., 1997). Due to matrix effects observed in the GC–MS method, calibration by
Fig. 2. LC–ESI-MS mass chromatograms, MS2 at m/z 288 for monitoring of AcABA: (A) blank urine; (B) urine of a rat dosed orally with 2 mg/kg of 2-NBA; (C) authentic 2-AcABA; (D) urine of a rat dosed orally with 2 mg/kg of 3-NBA; (E) authentic 3AcABA.
internal or external standard was not satisfactory. Therefore, standard addition method was used for quantification. A typical mass chromatogram is shown in Fig. 5. Results are shown in Table 1. For both isomers the total ABA excretion as determined by GC–MS is in agreement with the sum of free ABA and AcABA determined by LC–ESI-MS. Urine was collected in two subsequent 24 h intervals after dosing. Compared to the first 24-h post-exposure period, only 5–10% of these amounts were excreted in the second 24-h post-exposure period. This indicates that low conversion to these expected metabolites observed during the first day cannot be explained by delayed metabolism or excretion. 3.4. Excretion of 3-ABA-MA Mercapturic acid identified in the urine of rats dosed with 3-NBA was quantified by LC–ESI-MS using authentic standard. Excretion of this metabolite amounted less than 1% of the dose. The main portion of it was excreted during the first 24 h post-exposure period, only a minor portion was found in urine collected between 24 and 48 h after dosing (Table 1). 4. Discussion The carcinogenic 3-NBA differs from its isomers including 2NBA in many ways. Quantum chemical calculations have shown that the nitrenium ion derived from 3-NBA is the most stable of all benzanthronylnitrenium ions. Moreover, significant positive correlation was found between nitrenium ion stabilities of four NBA isomers and their mutagenic activities in Salmonella (Reynisson et al., 2008). In general, nitrenium ion stability is considered to be an important factor in mutagenicity and carcinogenicity of the corresponding parent compound (nitroarene or arylamine) because long living reactive intermediates can more easily reach their targets in nuclear DNA (Borosky, 2007). However, the calculated difference of
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Table 1 Excretion of urinary metabolites in rats after a single oral dose (2 mg/kg) of 2-NBA or 3-NBA. Total 2- and 3-ABA, i.e., sum of free plus conjugated forms were determined by GC–MS, other analyses were performed by LC–MS. Metabolite
Compound administered 2-NBA 2-ABA total
3-NBA 2-ABA free
Urinary excretion [% of dose; mean ± SE, n = 3] 0.32 ± 0.06 0.12 ± 0.05 0–24 h 24–48 h 0.008 ± 0.004 <0.01a a
2-AcABA
3-ABA total
3-ABA free
3-AcABA
3-ABA-MA
0.24 ± 0.11 <0.03a
0.83 ± 0.40 0.04 ± 0.01
0.10 ± 0.06 <0.01a
0.41 ± 0.25 <0.02a
0.49 ± 0.15 0.02 ± 0.01
Below the limit of quantification of the LC–ESI-MS method used, which ranged from 1.5 to 4 ng/mL for each analyte.
ca. 9 kcal/mol in the nitrenium ion energies of 2- and 3-NBA is very unlikely to account for the whole difference in their mutagenic and carcinogenic potentials. Our experiments on palladium catalysed reduction of 2- and 3NBA showed a markedly different reactivity, 3-NBA being much more reactive. In contrast, metabolic yield of total 2-ABA was only slightly lower than that of 3-ABA. In vivo, reductive metabolic activation of 3-NBA is catalysed mainly by NQO1 (Arlt et al., 2005; Stiborová et al., 2006). Despite strong binding of both 2- and 3-NBA to this enzyme, only 3-NBA seems to be efficiently metabolically activated to the corresponding nitrenium ion. This was demonstrated by strikingly different extent of DNA adduct formation both in vitro and in vivo (Arlt et al., 2007). Convincing explanation of this paradox was provided by Stiborová et al. Molecular docking of the two isomers into the active site of NQO1 showed similar
binding affinities but, unlike for 3-NBA, orientation of the 2-NBA molecule in the enzyme-substrate complex does not allow reduction of its nitro group, so that metabolic activation of 2-NBA by this enzyme cannot occur (Stiborová et al., 2010). This seems to be the key difference between metabolism of 2-NBA and 3-NBA, responsible for their strikingly different biological activities as well as for the lack of formation of 2-ABA-MA by biotransformation of 2-NBA. It is most likely that the reduction of 2-NBA to 2-ABA is catalysed by other enzyme(s) than NQO1, and the ultimate electrophilic intermediate, 2-benzantronylnitrenium ion, which is less stable than 3-benzanthronylnitrenium ion (Reynisson et al., 2008), is formed in a much smaller extent during this biotransformation. This explanation is supported also by in vitro DNA binding studies. In various human cells binding of 2-NBA was up to 14-fold lower than that of 3-NBA, as determined by 32 P-postlabelling (Arlt et al., 2007). In the present study we observed a marked difference between 2- and 3-NBA in the mercapturic acid formation, with only the carcinogenic 3-NBA being demonstrated as a mercapturic acid precursor. Mercapturic acids are formed by glutathione conjugation of electrophilic species, very often reactive metabolites of various xenobiotics. Therefore, formation of these detoxication products is
Fig. 3. LC–ESI-MS mass chromatograms, MS2 at m/z 407 for monitoring of 3-ABAMA: (A) blank urine; (B) urine of a rat dosed with 2 mg/kg of 3-NBA; (C) blank urine spiked with authentic 3-ABA-MA. MS2 of 3-ABA-MA is shown in the insert.
Fig. 4. LC–ESI-MS mass chromatograms, MS2 at m/z 407 for monitoring of 2-ABAMA: (A) blank urine; (B) urine of a rat dosed with 2 mg/kg of 2-NBA; (C) blank urine spiked with authentic 2-ABA-MA. MS2 of 2-ABA-MA is shown in the insert.
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5. Conclusion The observed difference between 2- and 3-NBA in the mercapturic acid formation in rats in vivo reflects differences in the reductive bioactivation pathway, which may be critical for their mutagenic and carcinogenic potential. If confirmed also in humans, 3-ABA-MA would be a promising biomarker of exposure to and effective dose of 3-NBA. Conflict of interest None. Acknowledgements Financial support of this work by Grant Nos. 2B08051 and MSM 604 613 73 01 from the Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged. We are also grateful to Mr. Jiˇrí Kosina for the HRMS measurement. References
Fig. 5. GC–MS determination of 3-ABA in the acid hydrolyzed rat urine: (A) control urine; (B) urine of a rat dosed orally with 3-NBA at 2 mg/kg; (C) urine of a rat dosed with 3-NBA, spiked with 3-ABA, 0.1 g/mL. 3-ABA was measured as a HFB derivative at m/z 441.
closely related to toxicity of the corresponding xenobiotic, particularly to its mutagenic and carcinogenic potential. Thus, formation of 3-ABA-MA seems to reflect strong mutagenic and carcinogenic potential of 3-NBA, which is in fact much stronger than that of 2-NBA. Regardless of the expected common formation of mercapturic acids derived from mutagenic nitroarenes or arylamines, these in fact appear to be either exceptional or mostly unexplored. To our knowledge, since the early studies of Boyland et al. (1962, 1963) mercapturic acid formation from any arylnitrenium ion has not been reported. Thus, our finding of 3-ABA-MA seems to be the first report of an arylnitrenium derived mercapturic acid formation in vivo after 48 years. Taking into account the toxicological relevance of mercapturic acid pathway our observation indicates that there is a need for further investigation of possible mercapturic acid formation from other mutagenic and/or carcinogenic nitroaromatics and aromatic amines. In the case of 3-NBA, the identified mercapturic acid seems to be a promising specific biomarker of exposure and a valuable surrogate biomarker of effective dose. It remains to be established whether or not it is formed also in humans. Urinary metabolites detected in this study account for only a minor portion of the dose. No other significant metabolites have been detected by the methods used. Further studies are needed to describe the overall metabolic fate of 2- and 3-NBA. Such studies would be greatly facilitated by the administration of radiolabelled substrates.
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