Redox Cycling in the Metabolism of the Environmental Pollutant and

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Redox Cycling in the Metabolism of the Environmental Pollutant and Suspected Human Carcinogen o-Anisidine by Rat and Rabbit Hepatic Microsomes† Karel Naiman,‡ Helena Dracˇ´ınska´,‡ Marke´ta Martıńkova´,‡ Miroslav Sˇulc,‡ Martin Dracˇ´ınsky´,§ Lucie Kejı´kova´,‡ Petr Hodek,‡ Jirı´ Hudecˇek,‡ Jirı´ Liberda,‡ Heinz H. Schmeiser,| Eva Frei,| and Marie Stiborova´*,‡ Department of Biochemistry, Faculty of Science, Charles UniVersity, AlbertoV 2030, 128 40 Prague 2, Czech Republic, Institut of Organic Chemistry and Biochemistry, V.V.i., Academy of Sciences, FlemingoVo n. 6, 166 00 Prague 6, Czech Republic, and Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ReceiVed March 21, 2008

We investigated the ability of hepatic microsomes from rat and rabbit to metabolize 2-methoxyaniline (o-anisidine), an industrial and environmental pollutant and a bladder carcinogen for rodents. Using HPLC combined with electrospray tandem mass spectrometry, we determined that o-anisidine is oxidized by microsomes of both species to N-(2-methoxyphenyl)hydroxylamine, o-aminophenol, and one additional metabolite, the exact structure of which has not been identified as yet. N-(2-Methoxyphenyl)hydroxylamine is either further oxidized to 2-methoxynitrosobenzene (o-nitrosoanisole) or reduced to parental o-anisidine, which can be oxidized again to produce o-aminophenol. To define the role of microsomal cytochromes P450 (P450) in o-anisidine metabolism, we investigated the modulation of this metabolism by specific inducers and selective inhibitors of these enzymes. The results of the studies suggest that o-anisidine is a promiscuous substrate of P450s of rat and rabbit liver; because P450s of 1A, 2B, 2E, and 3A subfamilies metabolize o-anisidine in hepatic microsomes of both studied species. Using purified enzymes of rat and rabbit (P450s 1A1, 1A2, 2B2, 2B4, 2E1, 2C3, 3A1, and 3A6), reconstituted with NADPH:P450 reductase, the ability of P450s 1A1, 1A2, 2B2, 2B4, 2E1, and 3A6 to metabolize o-anisidine was confirmed. In the reconstituted P450 system, rabbit P450 2E1 was the most efficient enzyme metabolizing o-anisidine. The data demonstrate the participation of different rat and rabbit P450s in o-anisidine metabolism and indicate that both experimental animal species might serve as suitable models to mimic the fate of o-anisidine in human. Introduction 2-Methoxyaniline (o-anisidine) is a potent carcinogen, causing tumors of the urinary bladder in both genders of F344 rats and B6C3F1 mice (1, 2). The International Agency for Research on Cancer (IARC) has classified o-anisidine as a group 2B carcinogen (2), which is possibly carcinogenic to humans. Besides its carcinogenicity, it exhibits other toxic effects, including hematological changes, anemia, and nephrotoxicity (1, 2). o-Anisidine is used as an intermediate in the manufacturing of a number of azo and naphthol pigments and dyes, which are used for printing (90%) and for paper (3%) and textile (7%) dyeing (1, 3). Such a wide use of this aromatic amine could result in occupational exposure. Furthermore, it may be released from textiles and leather goods colored with these azo dyes, and a large part of the population may be exposed. This carcinogen is also a constituent of cigarette smoke (2, 4). This strongly suggests that o-anisidine ranks not only among occupational pollutants produced in the manufacturing of chemicals, but also among environmental pollutants; it can be assumed †

This work is dedicated to Professor Dr. Marie Ticha´. * To whom correspondence should be addressed. Phone: +420-221951285. Fax: +420-2-21951283. E-mail address: stiborov@ natur.cuni.cz. ‡ Charles University. § Academy of Sciences. | German Cancer Research Center.

that human exposure is widespread. Indeed, o-anisidine was found in human urine samples in the general population, in concentrations of 0.22 µg/L (median) (5). In addition, hemoglobin adducts of o-anisidine were detected in blood samples of persons living in urban or rural areas of Germany (6–8). The adducts as well as o-anisidine in urine might originate not only from the sources mentioned above but also from a possible o-anisidine precursor, o-nitroanisol. This chemical was released into the environment in the course of an accident in a German chemical plant, subsequently causing local and regional contamination (6, 9, 10). In spite of potent rodent carcinogenicity, o-anisidine is weakly mutagenic (11–15). Its mutagenicity to Salmonella typhimurium (12–14) has been associated with both peroxidative activation and the involvement of N-acetyltransferases (16–20). The chemical induces weak chromosomal aberrations in Chinese hamster ovary cells (21), gene mutations in mouse lymphoma cells (22, 23) and intrachromosomal recombination in Saccharomyces cereVisiae (24). A statistically significant DNA damage in the urinary bladder of CD-1 mice exposed to o-anisidine determined by the single-cell gel electrophoresis (Comet) assay was detected (25). Moreover, Ashby and co-workers (17, 26) demonstrated that a weak, but significant, increase in the frequency of mutations was induced in urinary bladder in transgenic lacI (Big Blue) mice treated with this carcinogen. However, the chemical is negative in other in ViVo genotoxicity

10.1021/tx8001127 CCC: $40.75  2008 American Chemical Society Published on Web 07/15/2008

Metabolism of Carcinogenic o-Anisidine

assays, including the mouse micronucleus test and the DNA single-strand break assay in rat liver, spleen, kidney, and bladder (16, 27, 28). Recently, we found that o-anisidine is oxidatively activated by peroxidase and cytochrome P450 (P4501) to species binding to DNA in Vitro (29–32). We also demonstrated that o-anisidine forms DNA adducts in ViVo. The same adducts as found in DNA incubated with o-anisidine and human microsomes in Vitro were detected in urinary bladder, the target organ, and to a lesser extent, in the liver, kidney, and spleen of rats treated with o-anisidine (31). The o-anisidine-derived DNA adducts were identified as deoxyguanosine adducts formed from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine, which is generated by oxidation of o-anisidine with human and rat hepatic microsomes (31, 32). Preliminary experiments of Smith and Williams (33) showed that this carcinogen might also undergo O-demethylation that is P450-dependent and yields formaldehyde (34). The aim of the present work was to characterize the product(s) of this reaction and to identify structures of additional o-anisidine metabolites formed by microsomal P450s (31, 32). The participation of human P450 enzymes in o-anisidine oxidation has already been evaluated (31). P450 2E1 is believed to be the major human P450 catalyzing o-anisidine oxidation, although several other recombinant human P450s (1A2, 2B6, 1A1, 2A6, 2D6, and 3A4) have detectable activity (31). Comparison between human P450s and those of experimental animals is essential for the extrapolation of animal carcinogenicity and toxicity data to assess human health risk (35, 36). Hence, another aim of this study was to evaluate the efficiency of o-anisidine metabolism by hepatic P450s from species in which this agent is carcinogenic or toxic (rat and rabbit), and to compare the data with those generated in human enzymatic systems (31). In order to identify the rat and rabbit P450s capable of metabolizing o-anisidine, three experimental approaches were employed: (i) selective inhibition of P450s, (ii) induction of specific P450s, and (iii) utilization of the purified P450 reconstituted with NADPH:P450 reductase.

Experimental Procedures Caution: o-Anisidine is a potent carcinogen and should be handled with care. Wastes must be discarded according to appropriate safety procedures. Chemicals. Chemicals were obtained from the following sources: R-naphthoflavone (R-NF), β-naphthoflavone (β-NF), NADP+, NADPH, ketoconazole, diethyldithiocarbamate (DDTC), sulfaphenazole, 3-[(3-cholamidopropyl)dimethylammonio]-1propane sulfonate (CHAPS), dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, phosphatidylserine, glucose 6-phosphate, and chlorzoxazone from Sigma Chemical Co. (St. Louis, MO, USA); 7-pentoxyresorufin, 7-ethoxyresorufin, o-nitroanisole, o-anisidine, and o-aminophenol (>99% based on HPLC) from Fluka Chemie AG (Buchs, Switzerland); testosterone, 6βhydroxytestosterone from Merck (Darmstadt, Germany); glucose 6-phosphate dehydrogenase from Serva (Heidelberg, Germany); 1 Abbreviations: R-NF, R-naphthoflavone; β-NF, β-naphthoflavone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate; CID, collision-induced dissociation; P450, cytochrome P450; EI, electron impact; DDTC, sodium diethyldithiocarbamate; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid; HPLC, high-performance liquid chromatography; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; 3-IPMDIA, 3-isopropenyl-3-methyldiamantane; MS, mass spectrometry; M1, metabolite 1; M2, metabolite 2; PB, phenobarbital (5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)trion); PCN, pregnenolone-16R-carbonitrile (3β-hydroxy-20-oxopregn-5ene-16R-carbonitrile); RIF, rifampicin; r.t., retention time; TLC, thin layer chromatography.

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bufuralol and its 1′-hydroxyderivative were from Gentest Corp. (Woburn, MA, USA); glutathione from Roche Diagnostics Mannheim (Germany); and bicinchoninic acid from Pierce (Rockford, IL, USA). All these and other chemicals were of analytical purity or better. 2-Methoxynitrosobenzene was synthesized in analogy to the synthesis described earlier (37) by oxidation of N-(2-methoxyphenyl)hydroxylamine with potassium dichromate in water and identified by 1H NMR recorded on a 400 MHz instrument in CDCl3 (referenced to TMS): 7.68 (1H, m), 7.36 (1H, m), 6.86 (1H, m), 6.30 (1H, m), and 4.28 (3H, s). 3-Isopropenyl-3-methyldiamantane (3-IPMDIA) was synthesized according to Olah and collaborators (38). N-(2methoxyphenyl)hydroxylamine was synthesized by a procedure similar to that described earlier (39). Briefly, to a solution of 2 g of ammonium chloride and 90 mmol of o-nitroanisole in 60% ethanol/water, 180 mmol of zinc powder was added in small portions. After the addition of the first portion at room temperature, the reaction starts; this can be monitored by the rising temperature in the flask. The reaction mixture was kept at 10-15 °C using a cooling bath (ice/sodium chloride mixture) and slowly adding additional doses of zinc powder. After 1 h, excess zinc was removed by filtration, and ethanol was removed under reduced pressure. The product was extracted into 100 mL of ethyl acetate and crystallized by adding hexane. The yield was 60%. N-(2-Methoxyphenyl)hydroxylamine authenticity was confirmed by electrospray mass and CID spectra and high field proton NMR spectroscopy. The positive-ion electrospray mass spectrum exhibited the protonated molecule at m/z 140.1, while the CID of its ion fragments were at m/z 125.2, 108.1, and 109.1. The 1H NMR spectra were recorded at 400 MHz in dimethyl sulfoxide-d6. The central line of dimethyl sulfoxide at 2.500 ppm was used as the reference line. The spectra showed the presence of the following protons: 8.28 (1H, d, J ) 2.3 Hz, exchanged with CD3OD), 7.64 (1H, d, J ) 1.5 Hz, exchanged with CD3OD), 7.01 (1H, m, Σ J ) 9.6 Hz), 6.84 (2H, m, Σ J ) 15.0 Hz), 6.75 (1H, m, Σ J ) 16.9 Hz), and 3.75 (3H, s). o-Aminophenol authenticity was confirmed by ESI mass spectra and 1H and 13C NMR spectroscopy. The positive-ion massspectrum exhibited the protonated molecule at m/z 110.4, while the negative-ion mass spectrum the molecule at m/z 108.4. The NMR spectra were recorded on a Bruker Avance II-500 instrument (499.8 MHz for 1H and 125.7 MHz for 13C) in dimethyl sulfoxide-d6 and referenced to the solvent signal (δ 2.50 and 39.70, respectively). The 1H NMR spectrum showed the presence of the following signals: 8.96 (1H, bs), 6.65 (1H, m), 6.59 (1H, m), 6.54 (1H, m), 6.40 (1H, m), and 4.46 (2H, bs). The 13C NMR spectrum showed the presence of six signals of aromatic carbons: 144.26 (s), 136.78 (s), 119.80 (d), 116.75 (d), 114.73 (d), and 114.64 (d). Animal Experiments, Preparation of Microsomes, and Assays. The study was conducted in accordance with the Regulations for the Care and Use of Laboratory Animals (311/ 1997, Ministry of Agriculture, Czech Republic), which complies with the Declaration of Helsinki. Microsomes from the livers of 10 untreated rats and 3 rabbits were prepared by the procedure described previously (40). Microsomes from the livers of 10 male Wistar rats or 3 male rabbits pretreated with β-NF (40) were isolated as described (40, 41): those pretreated with phenobarbital (PB) were isolated as reported by Hodek et al. (42), those pretreated with pregnenolone-16R-carbonitrile (PCN) for rats (43) and rifampicin (RIF) for rabbits (44) were isolated as reported by Gut et al. (43) and Borek-Dohalska et al. (44), respectively, and those pretreated with ethanol or acetone were isolated using a procedure described by Yang et al. (45). Protein

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Figure 1. HPLC elution profiles of metabolites of o-anisidine. (A) 1 mM and (B) 0.25 mM o-anisidine incubated with rabbit microsomes. (C) 1.0 mM and (D) 0.25 mM N-(2-methoxyphenyl)hydroxylamine incubated with rabbit hepatic microsomes. (E) Synthetic N-(2-methoxyphenyl)hydroxylamine and o-anisidine. (F) o-Aminophenol. (G) o-Nitrosoanisole. For incubation conditions, see Experimental Procedures. Peaks eluting between 2.0 and 5.5 min, solvent front, NADPH and protein components of microsomes and NADPH-generation system.

concentrations in the microsomal fractions were assessed using the bicinchoninic acid protein assay with bovine serum albumin as a standard (46). The concentration of P450 was estimated according to Omura and Sato (47) by measuring the absorption

of the complex of reduced P450 with carbon monoxide. Rat and rabbit liver microsomes contained 0.6 and 1.8 nmol P450/ mg protein, respectively. Hepatic microsomes of rats induced with β-NF, PB, PCN, and ethanol contained 1.3, 2.7, 1.6, and



Figure 2. MS/MS spectrum of o-anisidine metabolite N-(2-methoxyphenyl)hydroxylamine (A) and EI-MS (70 eV) analysis of this metabolite (B). Structures of the assigned fragments are shown in (A). The ion at m/z 139.1 in (B) indicates the molecular ion of hydroxylated o-anisidine.

1.8 nmol P450/mg protein, respectively. Hepatic microsomes of rabbits induced with β-NF, PB, RIF, and acetone contained 3.6, 4.7, 3.7, and 2.2 nmol P450/mg protein, respectively. The activity of NADPH:P450 reductase in rat and rabbit hepatic microsomes was measured according to Sottocasa et al. (48) using cytochrome c as the substrate (i.e., as NADPH:cytochrome c reductase). NADPH:P450 reductase activities in hepatic microsomes of control (uninduced) rats and those induced with β-NF, PB, PCN, and ethanol were 0.210, 0.199, 0.325, 0.400, and 0.201 µmol/min/mg protein, respectively. The activities of this reductase in hepatic microsomes of control (uninduced) rabbits and those induced with β-NF, PB, RIF, and acetone were 0.200, 0.170, 0.250, 0.200, and 0.195 µmol/min/mg protein, respectively. Isolation of Individual P450s. P450 1A2, 2B4, 2C3, and 2E1 enzymes were isolated from liver microsomes of rabbits induced with β-NF (P450 1A2), PB (P450 2B4, 2C3), or ethanol (P450 2E1), by procedures described by Haugen and Coon (49) and Yang et al. (45). P450 3A1 and 3A6 were isolated from hepatic microsomes of rats and rabbits induced with PCN (43) and RIF (44), respectively. The procedure was analogous to that used for the isolation of P450 2B4. Rat P450 2B2 was isolated from liver microsomes of rats pretreated with PB by the procedure as described (50). Recombinant rat P450 1A1

protein was purified to homogeneity by the procedure described previously (51) from membranes of Escherichia coli transfected with a modified P450 1A1 cDNA. Rabbit liver NADPH:P450 reductase was purified as described (52). Rabbit liver Cytochrome b5 was prepared as described elsewhere (53). Incubations. Unless stated otherwise, incubation mixtures used for study of o-anisidine metabolism contained the following concentrations in a final volume of 100 µL of 100 mM sodium phosphate buffer (pH 7.4), 1 mM NADP+, 10 mM D-glucose 6-phosphate, 1 U/mL D-glucose 6-phosphate dehydrogenase (NADPH-generation system), a rat or rabbit hepatic microsomal fraction containing 0.04-1.0 nmol P450, and 0.1-2.0 mM o-anisidine dissolved in 1.0 µL of methanol. The reaction was initiated by adding the substrate. Purified P450 was reconstituted with NADPH:P450 reductase and cytochrome b5 (0.5 µM P450, 0.5 µM NADPH:P450 reductase, 0.5 µM Cytochrome b5, 0.5 µg/µL CHAPS, 2.0 µg/µL liposomes [dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, and phosphatidylserine (1: 1:1)], 3 mM reduced glutathione and 50 mM HEPES/KOH, pH 7.4) (54, 55). Aliquot of the mixtures containing 50-200 pmol of P450 were then added to the incubation mixtures. After incubation in open glass tubes (37 °C, 30 min), the reactions were terminated by adding 100 µL of methanol and centrifuged at 5,000g for 5 min. Metabolism of o-anisidine with rat and

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Figure 3. The positive- and negative-ion mass spectra of o-anisidine metabolite M2 (A) and o-aminophenol (B). The protonated ion at m/z 110.4 and the negative-ion at m/z 108.4 indicate the molecular mass of o-aminophenol.

Figure 4. Atmospheric pressure chemical ionization (APCI) mass spectrum of o-anisidine metabolite M1. The ion at m/z 122.8 indicates the molecular mass of the nitrenium/carbenium ion of o-anisidine.

rabbit P450 enzymatic systems was linear until 40 min. The supernatants were collected and 20 µL of aliquots applied onto a high-performance liquid chromatography (HPLC) column, where the metabolites of o-anisidine were separated. The HPLC was performed on a C-18 reversed-phase column (250 × 4.6 mm, 5 µm, Nucleosil 100-5, Macherey-Nagel, Duren, Germany). Metabolites were eluted with 18% methanol, 82% 7.18 µM aqueous ammonia, pH 8.0 (v/v), at a flow rate of 0.6 mL/ min and monitored at 254 nm. A methanol concentration of 1% (v/v) was used to dissolve o-anisidine added into incubations (see above). Methanol is known to be a competitive substrate for P450 2E1 and, at a 1% concentration, inhibits chlorzoxazone 6-hydroxylation catalyzed by human hepatic microsomes (56).

Therefore, the effect of this compound on the oxidation of o-anisidine, used as the HCl salt (17), by hepatic microsomes of rats and rabbits induced with ethanol and acetone, respectively, and on o-anisidine oxidation catalyzed by P450 2E1 reconstituted with NADPH:P450 reductase in the presence of cytochrome b5 was evaluated. In these cases, when 1 µL methanol was added into the incubations, no significant decrease in the levels of o-anisidine metabolites was detected. To characterize o-anisidine metabolites, fractions containing the metabolites were collected from multiple HPLC runs, concentrated on a speed-vac evaporator and analyzed by mass spectrometry. In addition to HPLC, thin-layer chromatography (TLC) on silica gel was also employed. In this case, the reaction



Table 1. o-Anisidine Metabolism in Rat (A) and Rabbit (B) Hepatic Microsomes Induced with Different Agents (A) o-anisidine metabolitesa hepatic microsomes from rats pretreated withb

M1

o-aminophenol

none - control microsomes β-naphthoflavone (P450 1A1/2) phenobarbital (P450 2B1/2) ethanol (P450 2E1) PCN (P450 3A)

26.5 ( 2.7 48.0 ( 4.8 52.5 ( 5.3 14.2 ( 1.3 37.4 ( 3.8

52.0 ( 5.1 68.9 ( 6.2 114.1 ( 9.1 41.9 ( 4.2 52.3 ( 5.2

(B) o-anisidine metabolitesa hepatic microsomes from rabbits pretreated withb

M1

none - control microsomes β-naphthoflavone (P450 1A1/2) phenobarbital (P450 2B4) aceton (P450 2E1) RIF (P450 3A6)

96.4 ( 9.2 69.8 ( 7.1 121.1 ( 12.1 89.7 ( 8.8 96.0 ( 9.5

o-aminophenol 69.5 ( 7.0 44.3 ( 3.2 65.3 ( 6.5 57.0 ( 5.8 71.3 ( 7.0

a The numbers are the peak area/min/nmol P450 for each metabolite; averages ( SEM of three determinations in separate experiments are shown. b Isoforms of P450 induced are shown in parentheses.

was stopped by adding ethyl acetate (2 × 100 µL) and the metabolites extracted. The ethyl acetate extracts were evaporated to dryness, residues dissolved in 50 µL of methanol, applied onto the plates of thin layer of silica gel, and developed in the solvent system consisting of ethyl acetate:petrol ether (2:3, v/v). Bands of interest (UV detection at 254 nm) were scraped from the plates, eluted with ethanol, concentrated on a speed-vac evaporator, and analyzed by mass spectrometry (MS). The metabolites were also identified by comparing their chromatographic properties on HPLC with those of synthetic standards; 2-methoxynitrosobenzene, o-aminophenol, N-(2-methoxyphenyl)hydroxylamine, and 2-methoxynitrobenzene were eluted at retention times (r.t.) of 8.8, 11.3, 19.7, and 57.5 min, respectively. Another aliquot of the reconstituted mixture was used to estimate P450 activities with typical substrates: 7-ethoxyresorufin O-deethylation (P450 1A1/2), coumarin 7-hydroxylation (P450 2A6), 7-pentoxyresorufin O-depentylation (P450 2B6) (57), tolbutamide methyl hydroxylation (P450 2C), bufuralol 1′-hydroxylation (P450 2D6), chlorzoxazone 6-hydroxylation (P450 2E1), and testosterone 6β-hydroxylation (P450 3A) ( (58) and references therein). In the control incubation, P450 was omitted from the reconstitution mixture. To study the metabolism of N-(2-methoxyphenyl)hydroxylamine, incubation mixtures contained the following concentrations in a final volume of 100 µL: 100 mM sodium phosphate buffer (pH 7.4), 1 mM NADP+, 10 mM D-glucose 6-phosphate, 1 U/mL D-glucose 6-phosphate dehydrogenase (NADPHgeneration system), a rat or rabbit hepatic microsomal fraction containing 0.04-1.0 nmol P450, and 0.1-1.0 mM N-(2methoxyphenyl)hydroxylamine dissolved in 1.0 µL of methanol. The reaction was initiated by adding the substrate. In parallel incubations, N-(2-methoxyphenyl)hydroxylamine was dissolved in distilled water instead of methanol. No significant differences in the oxidation of N-(2-methoxyphenyl)hydroxylamine were found between incubations with or without methanol as solvent. Mass Spectrometry. Positive- and negative-ion ESI mass spectra were recorded on a Finnigan LCQ-DECA quadrupole ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Metabolites (final concentration 1 pmol/µL) dissolved in methanol/water (1: 1, v/v) were continuously infused through a capillary held at 1.8 kV into the dynamic Finnigan nanoelec-

trospray ion source via a linear syringe pump (Harvard Apparatus Model 22) at a rate of 1 µL/min. The ionizer and ion transfer optics parameters of the ion trap were as follows: spray voltage, 1800 V; capillary temperature, 150 °C; capillary voltage, 14 V; tube lens offset, -22 V; octapole 1 offset, -7.4 V; lens voltage, -16 V; octapole 2 offset, -11.3 V; octapole r.f. amplitude, 450 V peak-to-peak (pp); and entrance lens voltage, -66.9 V. Helium was introduced at a pressure of 0.1 Pa to improve the trapping efficiency of the sample ions. The spectra were scanned in the range m/z 50-800 and the gating time was set to accumulate and trap 1 × 107 ions. The mass isolation window for precursor ion selection was set to 2 amu and centered on the 12C isotope of the pertinent ion. The background helium gas served as the collision gas for the collision-induced dissociation (CID) experiment. The relative activation amplitude was 35%, and the activation time was 30 ms. No broadband excitations were applied. Metabolites were also characterized by the standard EI (electron impact)-MS (70 eV) (FinniganMAT, San Jose, CA, USA). Inhibition Studies. The following inhibitors were used with o-anisidine in rat and rabbit hepatic microsomes: R-NF, which inhibits P450 1A1 and 1A2; furafylline, which inhibits P450 1A2; 3-IPMDIA, which inhibits P450 2B (50, 59); sulfaphenazole, which inhibits P450 2C; quinidine, which inhibits P450 2D; DDTC, which inhibits P450 2E1; and ketoconazole and troleandomycin, which inhibit P450 3A. Inhibitors were dissolved in 1.0 µL of methanol to yield final concentrations of 1-1000 µM in the incubation mixture. The complete mixtures without o-anisidine were then incubated at 37 °C for 5 min, o-anisidine was added, and incubation continued for a further 30 min at 37 °C. An equal volume of pure methanol was added to the control incubations.

Results Metabolism of o-Anisidine by Rat and Rabbit Hepatic Microsomes. When o-anisidine (1 mM) was incubated with rat and rabbit hepatic microsomes in the presence of NADPH, three product peaks with r.t. of 7.0, 11.3, and 19.7 min were separated by HPLC with UV monitoring at 254 nm (see peaks in Figure 1A for the profile obtained with rabbit microsomes). If lower concentrations of o-anisidine in incubations were used, no metabolite peak with r.t. of 19.7 min was detected by HPLC (see Figure 1B for 0.25 mM o-anisidine). The o-anisidine metabolites were characterized by mass spectrometry and/or by comparison of their chromatographic properties with those of authentic standards. The cochromatography of the product peak with r.t. of 19.7 min with N-(2-methoxyphenyl)hydroxylamine showed that their chromatographic properties were identical (Figure 1). Using mass spectrometry, the structure of this metabolite was confirmed. In the positive-ion electrospray massspectrum, this metabolite showed the protonated molecule at m/z 140.1 (Figure 2A), indicating the molecular mass of a hydroxylated derivative of methoxyaniline. The detection of a molecular peak at m/z 139.1 by an EI-MS (70 eV) technique confirmed that the compound is a hydroxyl derivative of o-anisidine (Figure 2B). The CID of the compound ion (Figure 2A) afforded a fragment at m/z 125.2 showing the mass difference equal to 15, representing a methyl group. Other fragments at m/z 108.1 and 109.1 show the molecular masses of protonated methoxybenzene and N-phenylhydroxylamine, respectively. Collectively, these results indicate that the analyzed metabolite is N-(2-methoxyphenyl)hydroxylamine. The chromatographic properties of an o-anisidine metabolite with a r.t. of 11.3 min (M2) corresponded to those of o-aminophenol

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Table 2. Inhibition Constants for Inhibitors of o-Anisidine Metabolism in Rat (A) and Rabbit (B) Hepatic Microsomes (A) IC50 (mM)c, for the formation of a

b

hepatic microsomes from rats pretreated with

inhibitor

R-naphthoflavone (P450 1A1/2) furafylline (p450 1a2) 3-IPMDIA (P450 2B) diethyldithio-carbamic acid (P450 2E1) sulfaphenazole (p450 2c) ketoconazole (p450 3a) troleandomycin (P450 3A)

β-naphthoflavone (P450 1A1/2) β-naphthoflavone (P450 1A1/2) phenobarbital (P450 2B1/2) ethanol (P450 2E1) control PCN (P450 3A) PCN (P450 3A)

M1 2.3d 3.5 0.05 n.i.f n.i. 3.6 n.i.f

o-aminophenol 2.1 4.0 0.01 n.i. n.i. 7.7 n.i.

(B) IC50 (mM)c, for the formation of a

b

hepatic microsomes from rats pretreated with β-naphthoflavone (P450 1A1/2) β-naphthoflavone (P450 1A1/2) phenobarbital (P450 2B4) acetone (p450 2E1) control rifampicine (P450 3A6) rifampicine (P450 3A6)

inhibitor

R-naphthoflavone (P450 1A1/2) Furafylline (P450 1A2) 3-IPMDIA (P450 2B) diethyldithio-carbamic acid (P450 2E1) sulfaphenazole (p450 2c) ketoconazole (p450 3a) troleandomycin (p450 3a)

M1 0.3d 1.0 0.025 1.1 n.i.f 2.1 7.5

o-aminophenol 0.4 1.2 0.04 0.1 n.i. 5.0 8.1

a Isoforms of P450 induced are shown in parentheses. b Isoforms of P450 inhibited are shown in parentheses. c Estimated from concentration-dependent inhibition of the formation of o-anisidine metabolites by interpolation (inhibitors were 0.001-10 mM depending on the chemical). o-Anisidine (100 µM) and 0.4 nmol of P450 were present in the incubation medium. d Averages of three determinations in separate experiments. f n.i., no inhibition, that is IC50 is greater than 10 mM.

Figure 5. Metabolism of o-anisidine by purified rat and rabbit P450s reconstituted with rabbit NADPH:P450 reductase. A 100 pmol amount of reconstituted P450/incubation and 0.1 mM o-anisidine were used in all experiments. Other conditions were as described in Figure 1. Values are averages and SEM of triplicate incubations. Rat P450 1A1, 2B2, and 3A1, and rabbit P450 1A2, 2B4, 2C3, and 3A6 were used in the reconstituted system.

(Figure 1). The mass spectra indicating the identity of this metabolite with o-aminophenol are shown in Figure 3. Chromatographic properties of an o-anisidine metabolite 1 (M1), eluting at r.t. of 7.0 min, did not correspond to any standard compound used for analysis. In the APCI (atmospheric pressure chemical ionization) mass spectrum, this metabolite showed the mass signal at m/z 122.8 (Figure 4), corresponding to that of the nitrenium/carbenium ion of o-anisidine. We have not ascertained whether this product is indeed the nitrenium/ carbenium ion metabolite of o-anisidine, formed in the incubation, or decomposition product formed in the mass spectrometer. No o-anisidine metabolism was observed with heat-inactivated microsomes or when NADPH was omitted from the incubation mixtures (data not shown). The results of additional experiments demonstrated that metabolites with r.t. of 7.0 and 11.3 min are also formed from N-(2-methoxyphenyl)hydroxylamine by microsomes (Figure 1C and D). When N-(2-methoxyphenyl)hydroxylamine was incu-

bated without hepatic microsomal enzymes or without NADPH, these metabolite peaks were also detectable by HPLC, but only under acidic conditions (pH 4.5 for 60 min). At pH 7.4, used for microsomal incubations, their spontaneous formation was negligible. During the metabolism of N-(2-methoxyphenyl)hydroxylamine by microsomes, a shoulder at 8.8 min was also detectable (Figure 1C and D), suggesting the formation of o-nitrosoanisole (2-methoxynitrosobenzene) (r.t. of 8.8 min, Figure 1G). Moreover, during the incubations of N-(2-methoxyphenyl)hydroxylamine with microsomes and NADPH, an additional product peak was detected by HPLC, being eluted with the same r.t. as the parental compound, o-anisidine (r.t. of 28.6 min) (Figure 1C and D). The mass spectra of this metabolite peak and o-anisidine were identical (not shown). Involvement of P450 Enzymes in o-Anisidine Metabolism in Rat and Rabbit Hepatic Microsomes. To quantitatively determine o-anisidine metabolism by rat and rabbit hepatic microsomes, formation of its two final metabolites, o-aminophenol and the metabolite 1, was evaluated. Therefore, o-anisidine was added to incubations at 0.1 mM concentration. Under such concentrations, the reactive metabolite of oanisidine, N-(2-methoxyphenyl)hydroxylamine, is not detectable because it is converted to o-aminophenol and metabolite 1. Incubations of o-anisidine with rat and rabbit microsomes isolated from the livers of uninduced animal models were carried out in the absence and/or presence of inhibitors of P450 enzymes, R-NF for P450 1A1/2, 3-IPMDIA for P450 2B, sulfaphenazole for P450 2C, quinidine for P450 2D, DDTC for P450 2E1, and ketoconazole for P450 3A. As expected, under the conditions used in the experiments, formation of oaminophenol and metabolite 1 was detected, while that of N-(2methoxyphenyl)hydroxylamine, because of its consumption by microsomal enzymes in incubations and/or its conversion to metabolite 1, was not detectable (Table 1). Metabolism of o-anisidine by hepatic microsomes of uninduced rats was inhibited by R-NF, 3-IPMDIA, DDTC, and ketoconazole, by 21%, 32%, 20%, and 20%, respectively. Likewise, metabolism of this carcinogen by hepatic microsomes of uninduced rabbits was inhibited by R-NF, 3-IPMDIA, DDTC, and ketoconazole,



Scheme 1. Pathways of o-Anisidine Metabolism by the Cytochrome P450 System Showing the Characterized Metabolites and Those Proposed to form DNA Adductsa

a

The compounds shown in brackets were not detected under the experimental conditions.

by 19%, 35%, 28%, and 26%, respectively. Sulfaphenazole and quinidine were ineffective inhibitors in the microsomes of both animals. These results suggest a role of P450 1A, 2B, 2E1, and 3A in o-anisidine metabolism by hepatic microsomes of uninduced rats and rabbits. It should be noted that the interpretation of the results of experiments with inhibitors is sometimes difficult because one inhibitor may be more effective with one substrate than another. Therefore, to confirm the role of these P450s in o-anisidine metabolism, the induction of individual P450 enzymes was performed with these animal models. Microsomes isolated from the livers of rats and rabbits pretreated with β-NF (enriched with P450 1A), PB (enriched with P450 2B), ethanol or acetone (enriched with P450 2E1), PCN for rats (enriched with P450 3A1/2), or RIF for rabbits (enriched with P450 3A6) were used (Table 1). While incubations of o-anisidine with the microsomes of rats pretreated with β-NF and PB led to a 1.5- and 2.1-fold increase (P < 0.05) in both M1 and o-aminophenol formation, respectively (Table 1A), inducers of other P450 enzymes had essentially no effect. Inhibitors of P450 1A, 2B and 3A, R-NF, 3-IPMDIA, and ketoconazole, respectively, inhibited o-anisidine metabolism by microsomes enriched with P450 1A1/2 (β-NFmicrosomes), P450 2B1/2 (PB-microsomes), and P450 3A (PCN-microsomes). A strong selective inhibitor of P450 2B, 3-IPMDIA (50, 59), was the most effective in inhibiting o-anisidine metabolism by hepatic microsomes of rats pretreated with PB with IC50 values of 10 and 50 µM for the formation of o-aminophenol and metabolite 1, respectively. An inhibitor of P450 1A1/2, R-NF, also decreased this reaction in hepatic microsomes of rats treated with β-NF, while furafylline, an inhibitor of P450 1A2, was less effective (Table 2A). Inhibitors of other P450 enzymes caused either weak (ketoconazole) or no inhibition (sulfaphenazole, DDTC, troleandomycin) (Table 2A). These results indicate that P450 2B, 1A and, to a lower extent, P450 3A are important in o-anisidine metabolism in rat livers. In contrast to rat microsomal systems, induction of P450 2B4 in rabbit livers had only low stimulation effect on the formation of the o-anisidine metabolite M1, but was without effect on the production of o-aminophenol. Treating rabbits with β-NF and

acetone seemed even to inhibit o-anisidine metabolism rather than inducing it (Table 1B). Induction of P450 3A6 in rabbit livers had practically no effect on the metabolism of o-anisidine (Table 1B). This suggests that none of the P450 enzymes expressed in rabbit livers plays a predominant role in o-anisidine metabolism, while all might, to some extent, participate in the reactions. Indeed, inhibitors of P450 1A1/2, 2B4, 2E1, and 3A6 decreased o-anisidine metabolism in hepatic microsomes of rabbits treated with inducers of the corresponding P450 enzymes, while sulfaphenazole, an inhibitor of P450 2C, was ineffective (Table 2B). The most efficient inhibitor of o-anisidine metabolism by hepatic microsomes of rabbits pretreated with inducers of the respective P450s was again 3-IPMDIA, followed by DDTC, R-NF, furafylline, ketoconazole, and troleandomycin. Therefore, P450 2B4, 2E1, 1A1/2, and 3A6 seem to be the enzymes metabolizing o-anisidine in the rabbit microsomal system. Nevertheless, because P450 inhibitors are not absolutely specific for individual P450s, the results found with them should be carefully interpreted. Metabolism of o-Anisidine by Purified P450 Enzymes. To further characterize the role of individual P450s in the metabolism of o-anisidine, several P450 enzymes were purified, reconstituted with NADPH:P450 reductase, and used as the enzymatic system. Because P450 2E1, 3A1, and 3A6 activities depend on the presence of cytochrome b5 (55), this protein was added to the mixture with these P450s and reductase. All reconstituted P450s were capable of oxidizing their typical substrates (data not shown), and most of them were also active with o-anisidine; o-aminophenol and o-anisidine metabolite 1 were detected. While among the P450 enzymes tested rabbit P450 2E1 was the most efficient enzyme metabolizing oanisidine, rat P450 1A1, 2B2, and rabbit P450 1A2, 2B4 and 3A6 were less active. Rat P450 3A1 and rabbit P450 2C3 were ineffective in the reconstituted system (Figure 5).

Discussion The results of this study show that rat and rabbit hepatic microsomes can metabolize carcinogenic o-anisidine. The hepatic microsomal P450 enzymes of these two animal species catalyze

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both O-demethylation of o-anisidine and its N-hydroxylation to form a reactive metabolite, N-(2-methoxyphenyl)hydroxylamine. This metabolite was found previously to generate deoxyguanosine adducts in DNA in Vitro (31). These adducts are also formed from o-anisidine after its oxidation by human hepatic microsomes and in ViVo, in rats treated with this carcinogen (31). The same deoxyguanosine adducts were also detected in the DNA of the urinary bladder, kidney, liver, and spleen of rats treated with o-nitroanisole (60), an oxidized counterpart of o-anisidine, and in DNA incubated with o-nitroanisole in Vitro with hepatic cytosolic enzymes and xanthine oxidase (60). These enzymatic systems were found to produce N-(2-methoxyphenyl)hydroxylamine after oanisidine reduction (61). The data indicate that the reactive N-(2methoxyphenyl)hydroxylamine formed as a metabolite of both carcinogens is critical for the formation of DNA lesions in target organs. Therefore, the P450 enzymes of liver microsomes of both species tested in this study participate in the activation pathway of o-anisidine. Using a combination of chromatographic and mass spectrometric methods, we were able to identify further o-anisidine metabolites, generated both from this compound and from its reactive metabolite, N-(2-methoxyphenyl)hydroxylamine. The results show that o-anisidine is a subject of complex redox cycling reactions. It is primarily oxidized to o-aminophenol and N-(2-methoxyphenyl)hydroxylamine. N-(2-methoxyphenyl)hydroxylamine is additionally converted to the nitrenium/ carbenium ion and/or a product whose mass spectrum corresponds to this ion. N-(2-methoxyphenyl)hydroxylamine is also oxidized to o-nitrosoanisole and reduced to the parent compound, o-anisidine. The formed o-anisidine may be O-demethylated again to o-aminophenol (Scheme 1). The question whether o-aminophenol is also formed from N-(2-methoxyphenyl)hydroxylamine by its O-demethylation to N-(2-hydroxyphenyl)hydroxylamine, which is subsequently reduced to o-aminophenol (Scheme 1), remains to be answered. No metabolites formed by this reaction were observed. The structure of the o-anisidine metabolic product eluting at 7.0 min (M1) remains unclear. Aromatic hydroxylamine derivatives are known to react with aromatic nitroso compounds forming azoxy compounds (62). We prepared two isomeric azoxy derivatives by reaction of N-(2-methoxyphenyl)hydroxylamine with o-nitrosoanisole (62). None of the prepared azoxy derivatives had similar chromatographic properties as the metabolite M1 (results not shown). Another possible reaction, which could occur, is a Bamberger rearrangement (63) of N-(2methoxyphenyl)hydroxylamine that would result in 4-amino3-methoxyphenol. This compound is unstable (64), and it is possible that it would give an ion with m/z 122 in mass spectra after protonation and loss of neutral water molecule. However, we did not have any standard of this compound, and thus, we could not evaluate whether the structure of M1 is 4-amino-3methoxyphenol. Recently, redox cycling reactions similar to those we found with o-anisidine were observed by Kim et al. (65), who studied metabolism of several aromatic and heterocyclic amines by a P450 1A2/NADPH:P450 reductase enzymatic system. They reported that the P450 system catalyzes oxidation of the N-hydroxylated intermediate formed from the carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to a nitrosoderivative. They demonstrated that NADPH: P450 reductase can catalyze the reduction of the IQ oxidation products, N-nitroso-IQ and N-hydroxyl-IQ, to N-hydroxyl-IQ and the parent amine, IQ (65). N-Hydroxylation products of two other aromatic amines investigated by Kim et al. (65), 2-aminofluorene and 4-aminobiphenyl, are, however, reduced

Naiman et al.

nonenzymatically by NADPH. We have not determined whether reduction of N-(2-methoxyphenyl)hydroxylamine to o-anisidine requires catalysis by NADPH:P450 reductase or occurs nonenzymatically. Such a study is under way in our laboratory. The present study documents the role of specific microsomal P450 enzymes in the metabolism of o-anisidine to o-aminophenol and the metabolite M1, which decomposes to a nitrenium/ carbenium ion during MS. The results indicate that o-anisidine is a promiscuous substrate of rat and rabbit hepatic P450 enzymes; P450s of 1A, 2B, 2E, and 3A subfamilies metabolize o-anisidine in hepatic microsomes of studied species. Using purified enzymes (P450 1A1, 1A2, 2B2, 2B4, 2E1, and 3A6) reconstituted with NADPH:P450 reductase, their ability to metabolize o-anisidine was confirmed. In the reconstituted P450 system, rabbit P450 2E1 was the most efficient enzyme metabolizing o-anisidine. Our experiments do not indicate which P450 enzymes preferentially catalyze O-demethylation versus N-hydroxylation. Since o-anisidine can be demethylated directly or via N-(2-methoxyphenyl)hydroxylamine, it was not possible to elucidate which of the enzymes preferentially demethylates which substrate. N-(2-Methoxyphenyl)hydroxylamine is a reactive compound, which can either easily decompose to the nitrenium/carbenium ion or serve as a substrate of P450s and probably also of NADPH:P450 reductase to form other products including o-aminophenol (Scheme 1). Further studies are planned to answer the questions concerning the participation of P450s and NADPH:P450 reductase in the formation of individual o-anisidine metabolites. The results of the present and former studies (31) show a similarity among P450s metabolizing o-anisidine in humans, rats, and rabbits. o-Anisidine was found to be a substrate for several human P450s, namely, of P450 1A2, 2E1, 2B6, 1A1, 2A6, 2D6, and 3A4 (31), and thus orthologous human and animal P450 enzymes are responsible for the metabolism of this carcinogen. This finding might be one of the criteria important to show that rodents might be suitable models to predict human metabolic susceptibility to o-anisidine. This is important in view of the evaluation of o-anisidine carcinogenicity as a carcinogenic risk factor for humans, particularly persons exposed to o-anisidine during azo dye production. The analysis of o-anisidine metabolites in the urine of such individuals as well as the determination of o-anisidine-derived DNA adducts in lymphocytes should be used to monitor these workers. Nevertheless, also other criteria, such as the type of tumor or organs of tumorigenesis, might be even more important to estimate o-anisidine carcinogenicity to human. Therefore, the extensive examination of these persons for the organ specific cancer development should also be monitored. While the formation of N-(2-methoxyphenyl)hydroxylamine was clearly identified to be the activation pathway of o-anisidine metabolism (31), biological significance of its O-demethylation to o-aminophenol for detoxication/activation metabolism awaits further investigation. o-Aminophenol might be considered to be mutagenic because it induces sister chromatid exchanges in a dose-dependent manner in cultured human lymphocytes in Vitro and in Chinese hamster bone marrow cells in ViVo (66). In addition, Brennan and Schiestl (67) reported that o-aminophenol is positive in the deletion recombination assay in Saccharomyces cereVisiae. Even though o-aminophenol has not been found to form covalent DNA adducts, it was demonstrated in in Vitro experiments to cause DNA damage, forming 8-oxy7,8-dihydro-2′-deoxyguanosine in the presence of metal ions such as Cu(II) (68). Hence, because of such processes, oaminophenol may contribute to the initiation of the carcino-


genesis mediated by o-anisidine in the urinary bladder and to the development of tumors induced by other bladder carcinogenic aromatic amines (69), which produce this compound as one of the metabolites (67, 69). Furthermore, O-demethylation reactions produce formaldehyde (Scheme 1), which is known to modify DNA, generating several products including hydroxymethyl adducts and cross-links (70–72). Formaldehyde is mutagenic in a variety of different test systems and carcinogenic in laboratory animals (73) and has been described as “carcinogenic to human” by the IARC and “reasonably anticipated to be a human carcinogen” by the U.S. Department of Health and Human Services (74, 75). Therefore, on the one hand, it is plausible that formaldehyde-DNA adducts could also play a role in carcinogenesis by o-anisidine. On the other hand, however, formaldehyde produced in the cell is also detoxified by conjugation to glutathione and oxidized (76); therefore, it is not likely a strong contributor to carcinogenicity by o-anisidine. It should be noted that tumor development in a specific organ is influenced by promotional pressures on initiated cells in target organs and not only by the levels of DNA adducts formed by compounds such as o-anisidine. It is known that radicals formed from several carcinogens producing oxidative DNA damage, such as 8-hydroxy-2′-deoxyguanosine, are important not only in initiation but also in the promotion phases of carcinogenesis (77). Therefore, their formation from o-aminophenol may be one of the factors contributing to tumor promotion in oanisidine-mediated carcinogenesis. In addition, o-anisidine is oxidized by several peroxidases, which are expressed in target organs (e.g., COX), to form radicals besides DNA adducts (29, 30). Hence, the production of such free radicals in or near the target cells may be another factor important in the promotional process in o-anisidine-mediated tumor development. However, the exact functions of such and/or other promotional pressures caused both by o-anisidine and o-aminophenol in an o-anisidine-mediated tumorigenesis remains to be resolved.


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(11) (12)

(13) (14)

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Acknowledgment. This research was supported in part by Grant Agency of Charles University (grant 7418/2007), Grant Agency of the Czech Republic (grant 203/06/0329), and the MinistryofEducationoftheCzechRepublic(grantMSM0021620808). We thank Dr. Martin Sˇtıćha for the mass spectrometric analyses.

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