Reduction of Aromatic and Heterocyclic Aromatic N ...courses.washington.edu/medch582/PDFs/2014_Feb10_Robert.pdfP450-catalyzed N-hydroxylation is usually involved as the initial step

Reduction of Aromatic and Heterocyclic Aromatic N‑Hydroxylaminesby Human Cytochrome P450 2S1Kai Wang and F. Peter Guengerich*

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, School of Medicine, 638 Robinson ResearchBuilding, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, United States

*S Supporting Information

ABSTRACT: Many aromatic amines and heterocyclicaromatic amines (HAAs) are known carcinogens for animals,and there is also strong evidence of some in human cancer.The activation of these compounds, including some arylaminedrugs, involves N-hydroxylation, usually by cytochrome P450enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). Wepreviously demonstrated that the bioactivation product of theanticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F.P. (2012) Chem. Res. Toxicol. 25, 1740−1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved inthe reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl(4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 underboth anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobicconditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yieldedamines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the aminestested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several ofthe activated products of carcinogenic aromatic amines and HAAs.

■ INTRODUCTION

P450 enzymes catalyze a variety of reactions and are ofsignificant importance in the areas of drug metabolism andtoxicology.1,2 On the basis of analyses of marketed drugs, ∼75%of drug metabolism reactions involve P450s; five P450s (1A2,2C9, 2C19, 2D6, and 3A4) account for ∼90% of the P450reactions.3,4 Another recent analysis indicated that 66% ofcarcinogens are bioactivated by P450s, and three Family 1P450s show dominant roles, involved in ∼50% of the totalactivations attributed to P450s (1A1, 20%; 1A2, 17%; 1B1,11%).5

Aromatic amines [arylamines (Scheme 1); e.g., 4-amino-biphenyl (4-ABP), 2-naphthylamine (2-NA), 2-aminofluorene(2-AF)] are important industrial intermediates for theproduction of azo dyes and complex chemicals and are alsoused as antioxidants in rubber manufacturing processes.6,7 Rehnfirst reported the association of exposure to aromatic amines in1895 with the incidence of bladder cancer among German andSwiss workers in aniline dye factories.8 Further studies haveshown that aromatic amines induce tumors at multiple sites inrodents, dogs, and other experimental laboratory animals aswell.6,7,9−11

Heterocyclic aromatic amines [HAAs (Scheme 1); e.g., 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)] are producedin grilled and charred meats.12,13 Several HAAs have also been

detected in beer, wine,14 and cigarette smoke condensate.15,16

The Maillard reaction is believed to be involved in theformation of the imidazo-containing HAAs when amino acidsand carbohydrates are heated together,17,18 and other HAAsmay form through the pyrolysis of amino acids and proteins.19

Extensive studies have shown that HAAs are highly genotoxicin bacterial and mammalian cells and exhibit strongcarcinogenic effects in experimental animals.20−23

The role of P450s in the bioactivation of aromatic aminesand HAAs has been well-documented24−26 and reviewed.7,13,27

P450-catalyzed N-hydroxylation is usually involved as the initialstep in the process of chemical carcinogenesis with suchcompounds (Scheme 2).26,28 The N-hydroxylamines can befurther activated to more reactive esters, by N-acetyltransferases(NAT), sulfotransferases (SULT), and other enzymes.7

Subsequently, the resulting ester breaks down to yield anextremely unstable intermediate, a nitrenium ion, which reactswith DNA predominantly at position C8 of guanine bases andpotentially causes DNA mutations (alternatively, the nitreniumion can rearrange, and the active molecule will react with a N2guanine atom).29 Family 1 P450 enzymes (1A1, 1A2, and 1B1)are responsible for the metabolic activation of many aromaticamines, HAAs, and polycyclic aromatic hydrocarbons.24,30−36

The expression of P450 genes in this family is regulated by the

Received: April 8, 2013Published: May 18, 2013

Article

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© 2013 American Chemical Society 993 dx.doi.org/10.1021/tx400139p | Chem. Res. Toxicol. 2013, 26, 993−1004

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aryl hydrocarbon receptor (AhR).37 P450 1A2 is expressedalmost exclusively in liver in humans (10−15% of total P450),38

but P450 1A1 and 1B1 are generally not present in liver butexpressed in extrahepatic tissues at various levels.39 A numberof in vitro and in vivo studies have shown that P450 1A2 plays amajor role in the bioactivation of aromatic amines and HAAs inrodents and humans.30,40,41

The human genome has 57 P450 genes. P450 2S1 is one of∼13 that are classified as “orphans” with only limited evidenceof function.42 P450 2S1 mRNA has been detected in liver, skin,tumors, and other tissues,43−46 and the gene is regulated by theAhR.47,48 The sites of expression and mode of regulationsuggest that P450 2S1 might be involved in the process ofchemical carcinogenesis.45 Recently, attenuation of P450 2S1by siRNA has been shown to enhance cell proliferation andmigration in bronchial epithelial cells.49 However, a number ofcarcinogens have been tested and shown not to be activated byP450 2S1.50 Retinoic acid was reported as a substrate for P4502S1,44 but subsequent efforts by others have failed to reproducethese results.51,52 The first repeatedly demonstrated reaction forP450 2S1 is the anaerobic reduction of a di-N-oxide anticancerprodrug, 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (AQ4N), to its amino deriv-ative, 1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dehydrox-yanthracene-9,10-dione (AQ4).51,52 Previously, we demonstra-ted that an N-hydroxylarylamine, 2-(4-hydroxylamino-3-meth-ylphenyl)-5-fluorobenzothiazole (HONH-5F 203), can bereduced to its amine precursor, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, an anticancer agent), by P4502S1 under anaerobic conditions.53

In this study, several N-hydroxylarylamines and nitroso andnitro derivatives of common carcinogenic aromatic amines andHAAs were synthesized and shown to be reduced by P450 2S1.The rates of P450 2S1-catalyzed anaerobic reductions of the N-hydroxylarylamines were determined. However, N-hydroxyphe-nacetin (the activated product of the analgesic drug

phenacetin54) was not reduced by P450 2S1. Our resultssuggest that P450 2S1 may be involved in the detoxication ofthe activated products of a number of aromatic amines andHAAs.

■ EXPERIMENTAL PROCEDURESCaution: Many of the compounds used in this study are carcinogens

and should be handled with care! Gloves, lab coats, and safety gogglesshould be worn when handling these compounds.All commercially obtained solvents and other chemicals were useddirectly without further purification. 4-Nitrophenetole, 1-amino-naphthalene, 2-aminonaphthalene, and 2-nitrofluorene were purchasedfrom Sigma-Aldrich (St. Louis, MO). 2-Nitronaphthalene, 4-nitro-biphenyl, NO2-IQ, NO2-MeIQx, and NO2-PhIP were kindly providedby Robert. J. Turesky (Wadsworth Center, Albany, NY). NMR datawere obtained with a 400 MHz Bruker NMR spectrometer, using thesolvents noted, at Vanderbilt. UV spectra were obtained (online) witha Waters Acquity UPLC system equipped with a photodiode arraydetector (Waters, Milford, MA).

Chemical Synthesis. N-Hydroxyphenacetin. The title compoundwas prepared using a modification of a previously reportedprocedure.54 4-Nitrophenetole (0.10 g, 0.60 mmol) was reduced toN-hydroxyphenetidine in 2 mL of a C2H5OH/H2O mixture (3:1, v/v)by Zn dust (0.16 g, 2.4 mmol) in the presence of 0.032 g of NH4Cl.The reaction mixture was diluted with 4 mL of cold H2O and extractedwith (C2H5)2O (3 × 5 mL). The combined (C2H5)2O extracts wereevaporated to dryness in vacuo at 23 °C. The residue was dissolved in 2mL of (C2H5)2O in the presence of a slurry of NaHCO3 (0.08 g in0.25 mL of water) at 0 °C, and 2.5% acetyl chloride (47 mg, 0.60mmol) in (C2H5)2O (w/v) was added slowly to the mixture. Theresulting mixture was stirred for 2 h and extracted with (C2H5)2O (3 ×5 mL). The combined (C2H5)2O layers were washed sequentially withH2O and brine and dried over anhydrous Na2SO4. The solvent wasremoved in vacuo, and flash column chromatography on silica gel using(C2H5)2O yielded N-hydroxyphenacetin as colorless crystals (0.053 g,45% yield from 4-nitrophenetole, mp 101−103 °C, lit.55 mp 103 °C):1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.8 Hz, aromatic, 2H),6.85 (d, J = 8.9 Hz, aromatic, 2H), 4.01 (q, J = 7.0 Hz, 2H, CH3CH2-),

Scheme 1. Structures of Carcinogenic Aromatic and Heterocyclic Aromatic Amines

Scheme 2. Biotransformation Pathways of Aromatic and Heterocyclic Aromatic Amines

Chemical Research in Toxicology Article

dx.doi.org/10.1021/tx400139p | Chem. Res. Toxicol. 2013, 26, 993−1004994

2.16 (s, 3H, CH3CO−), 1.40 (t, J = 7.0 Hz, 3H, CH3CH2−); GC−MS(electron impact) m/z 179, 137, 108.N-Hydroxylamines and Nitroso Compounds. HONH-5F 203,53 4-

HONH-biphenyl, 2-HONH-naphthalene, 2-HONH-fluorene, andHONH-PhIP were prepared by the reduction of the correspondingnitro derivatives with hydrazine and Pd/C at 0 °C for 30 min to 2h.56,57 HONH-IQ and HONH-MeIQx were prepared by the reductionof nitro analogues with ascorbic acid in (CH3)2SO as described byTuresky et al.57 NO-5F 203 and 4-NO-biphenyl were prepared by theoxidation of HONH-5F 203 and 4-HONH-biphenyl with K3Fe(CN)6under ambient conditions for 3 h as reported previously.53

Enzyme Preparations. Recombinant human P450 2S1 wasexpressed and purified in Escherichia coli membranes as previouslydescribed.50 NADPH-P450 reductase (NPR) was expressed in E. coliand purified as described previously.58

Incubation Conditions. A typical incubation59 mixture containedpurified P450 2S1 (0.5 μM), NPR (1 μM), 160 μM L-α-dilauroyl-sn-glycero-3-phosphocholine, an NADPH-generating system (10 mMglucose 6-phosphate, 0.5 mM NADP+, and 4 μg/mL yeast glucose-6-phosphate dehydrogenase), and the appropriate substrate (100 μM) in0.10 M sodium N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid(HEPES) buffer (pH 7.4, containing 1 mM EDTA). The incubationswere performed at 37 °C for 30 min. Cold CH2Cl2 (2-fold volume)was added to terminate each reaction, and the resulting mixtures wereseparated by centrifugation at 2000g for 3 min. The lower organic layerwas transferred and dried under a N2 stream. The residue wasdissolved in CH3CN for HPLC and LC−MS analysis.Anaerobic Incubations. Qualitative anaerobic experiments were

performed in Thunberg tubes, as previously described.53 A typical 500μL incubation mixture contained 0.5 μM P450, 1.0 μM NPR, 160 μML-α-1,2-dilauoryl-sn-glycero-3-phosphocholine, 100 mM sodiumHEPES buffer (pH 7.4), 1 mM EDTA, and 100 μM substrate [froma freshly prepared 10 mM (CH3)2SO stock]. When the desired level ofanaerobicity was achieved (after five cycles of vacuum and Ar), anNADPH-generating system was added to the incubation mixture fromthe neck of the tube. The reaction mixtures were incubated at 37 °Cfor 10−30 min, the reactions terminated by the addition of 1.0 mL ofcold CH3CN, and the mixtures analyzed by HPLC.For comparisons, anaerobic incubations were conducted with P450

1A1, 1A2, 2S1, 2W1, and 3A4 at the same concentration (0.2 μM) for10 min.Steady-State Kinetic Measurements of Anaerobic Reduction

of HONH-5F 203. Anaerobic incubations were conducted under a N2atmosphere in a glovebox (Labconco, Kansas City, MO). Theconcentrations of P450 and the NADPH-generating system and bufferconditions remained the same as those employed in the qualitativestudies. A stock solution was made of freshly prepared HONH-5F 203in (CH3)2SO, and the concentration was determined colorimetri-cally.60 A series of standard solutions at different concentrations wasprepared from the stock solution by dilution. Reactions with differentsubstrate concentrations (ranging from 0 to 200 μM) were conducted,in duplicate, at 37 °C for 10 min. After the reactions had beenquenched with CH2Cl2 (2-fold volume), 2-phenylbenzothiazole (5 μL,0.1 mM) was added to each reaction mixture as an internal standard.The mixtures were centrifuged at 2000g for 3 min, and the organiclayers were transferred and dried under a stream of N2. The residueswere dissolved in CH3CN for HPLC analysis.Determination of Rates of Anaerobic Reduction of 4-NHOH-

Biphenyl, 2-NHOH-Naphthalene, and 2-NHOH-Fluorene to theCorresponding Amines by P450 2S1. Anaerobic incubations wereconducted under a N2 atmosphere in a glovebox, as in the steady-statekinetic studies for HONH-5F 203 reduction. The rates of reductionwere measured at a single substrate concentration (10 μM). Standardsolutions (1 mM, determined by colorimetric assay) of 4-NHOH-biphenyl, 2-NHOH-naphthalene, and 2-NHOH-fluorene in CH3CNwere prepared from freshly prepared samples. Under the conditionsused in this study, optimal P450 2S1 concentrations for 4-NHOH-biphenyl, 2-NHOH-naphthalene, and 2-NHOH-fluorene were 500, 25,and 50 nM, respectively (the corresponding concentrations of NPRwere 1, 0.5, and 0.5 μM, respectively). The concentrations of the

NADPH-generating system and buffer conditions were the same as ina typical incubation, vide supra. A standard solution (5 μL) was addedto each enzyme reaction mixture (volume of 500 μL) and each mixtureincubated at 37 °C. Reactions were terminated by addition of CH2Cl2(1.0 mL) after 0, 1, 2, 3, 4, and 5 min. For each compound,incubations were performed in duplicate at every time point.Incubations devoid of P450 2S1 were performed as controls. 1-Naphthylamine (5 μL, 500 μM) was added to each sample as aninternal standard, and the mixtures were centrifuged. The isolatedorganic layers were transferred and dried under a stream of N2, and theresidues were dissolved in CH3CN for HPLC analysis.

HPLC and LC−MS Analysis of the Reduction Reactions. Thereduced reaction products of hydroxylamine, nitroso, and nitrocompounds were analyzed using a Waters Acquity UPLC systemequipped with a photodiode array detector (Waters) and anoctadecylsilane (C18) reversed-phase column (5 μm, 2.1 mm × 250mm, Hypersil GOLD, Thermo Scientific, Odessa, TX). Absorbancewas integrated (210−400 nm). The column temperature wasmaintained at 25 °C. LC conditions for phenacetin, 4-ABP, 2-NA,2-AF, and 5F 203 derivatives were as described previously.53 Solvent A[5:95 CH3CN/H2O mixture with 0.5% HCO2H (v/v/v)] and solventB [95:5 CH3CN/H2O mixture with 0.5% HCO2H (v/v)] were used.The gradient started from 5% B (v/v) during the first 2 min (0−2min), increased linearly to 100% B over 5 min (2−7 min), was held for4.5 min (7−11.5 min), and then returned to 5% B (v/v) over 0.5 min(11.5−12 min) before re-equilibration (12−15 min), at a flow rate of0.5 mL/min. LC conditions for the IQ, MeIQx, and PhIP derivativeswere as follows. Solvent A contained 20 mM NH4CH3CO2 and 5%CH3CN (v/v), and solvent B contained NH4CH3CO2 (20 mM, pH6.8) and 95% CH3CN (v/v). A linear gradient was employed with aflow rate of 0.5 mL/min, starting from 5% solvent B and reaching100% solvent B at 12 min, and the percentage of solvent B returned to5% (v/v) over 3 min (12−15 min).

LC−MS and LC−MS/MS analyses were performed on a WatersAcquity UPLC system coupled to a Thermo-Finnigan LTQ ion trapmass spectrometer (Thermo Fisher Scientific, San Jose, CA). Thesame LC conditions were used for each sample as in the HPLCanalysis. Samples were generally analyzed in the atmospheric-pressurechemical ionization (APCI) positive ion mode. MS conditions wereoptimized for each amine derivative as follows: capillary temperature,275 °C; vaporizer temperature, 450 °C; source voltage, 6.0 kV; sheathgas flow, 50.0; auxiliary gas flow, 5.0; sweep gas flow, 5.0; and in-sourcefragmentation, 10 V.

Colorimetric Assay for N-Hydroxyarylamines.60 From atypical solution containing 5−10 mM N-hydroxyarylamine, a 1−2μL aliquot was added to a tube containing 0.96 mL of the colorreagent.60 The sample was mixed vigorously using a vortex device.After 3 min, 40 μL of stabilizer (H3PO4, 20 mM) was added to themixture. The sample was mixed thoroughly, and A535 was measured,with reference to a sample containing all of the reagents except N-hydroxyarylamine as a control (ε535 = 39200 M−1 cm−1).

P450 2S1 Binding Assay (with HONH-5F 203). Binding spectraof P450 with HONH-5F 203 were recorded with purified P450 2S1(23 °C) using a UV−visible scanning spectrophotometer (AmincoDW-2a, OLIS, Bogart, GA). A baseline was recorded (350−700 nm)using two 1.0 mL glass cuvettes (1 cm path length) containing equalvolumes of 100 mM potassium phosphate buffer (pH 7.4) containing20% glycerol (v/v). Purified P450 2S1 was added to the samplecuvette (final concentration of 5 μM). The same volume of the bufferwas added to the reference cuvette. Equal amounts of freshly preparedaliquots of HONH-5F 203 [dissolved in (CH3)2SO] were added toboth cuvettes. Difference spectra were collected (350−700 nm) afteran equilibration period of 1 min [total volume of (CH3)2SO of ≤2%].

■ RESULTS

Search for Oxidations of Potential Substrates withP450 2S1. No detectable products were observed followingthe incubation of aniline, 7-ethoxycoumarin, IQ, MeIQx, orPhIP with human P450 2S1, in typical aerobic incubation



systems containing NPR and NADPH, as judged by HPLC−UV assays. No products were detected from an incubationmixture of [14C]lauric acid with P450 2S1 using HPLC(coupled online with scintillation counting).Anaerobic Incubation of N-Hydroxyphenacetin with

P450 2S1. N-Hydroxyphenacetin was incubated with P4502S1 at 37 °C for 20 min under anaerobic or aerobic conditions,with an HPLC peak at a tR of 4.7 min corresponding to N-hydroxyphenacetin. N-Hydroxyphenacetin was stable under allconditions tested in this study (Figure S1 of the SupportingInformation). Neither P450 2S1, NPR, NADPH, nor thecombination changed the N-hydroxyphenacetin to anyproducts.Anaerobic Reduction of 4-HONH-Biphenyl, 2-HONH-

Naphthalene, and 2-HONH-Fluorene with P450 2S1. N-Arylhydroxylamines are unstable substances and readily under-go disproportionation reactions (Scheme 2) to yield arylaminesand nitroso derivatives in neutral or basic media.61,62 Thenitroso products can further condense with N-arylhydroxyl-amines to produce azoxy compounds.63 In anaerobic

incubations of all three N-arylhydroxylamines with P450 2S1(in the presence of NPR and an NADPH-generating system),significantly larger amounts of the corresponding arylamineswere formed [than under other conditions when P450 2S1 oran NADPH-generating system was absent (Figure 1, andFigures S3 and S5 of the Supporting Information)], indicatingthat P450 2S1 reduced the N-arylhydroxylamines to arylamines(Scheme 2). Under aerobic incubation conditions (Figure S2 ofthe Supporting Information), competitive disproportionationswere favored that also yielded arylamines as products. Thereduction of N-arylhydroxylamines by P450 2S1 was lessapparent compared to what occurred under anaerobicconditions.HPLC analyses of the incubation of 4-HONH-biphenyl with

P450 2S1 showed four major peaks (Figure 1). 4-ABP (λmax =250 nm), 4-HONH-biphenyl (λmax = 273 nm), 4-NO-biphenyl(λmax = 345 nm), and azoxybiphenyl (λmax = 357 nm) wereeluted at tR values of 4.5, 5.5, 6.8, and 8.2 min, respectively. 4-Nitrobiphenyl (λmax = 310 nm) appeared at a tR of 6.6 minunder the same HPLC conditions (data not shown). 4-ABP [tR

Figure 1. Anaerobic incubations of 4-HONH-biphenyl with P450 2S1. Freshly prepared 4-HONH-biphenyl [5 μL of a 10 mM solution in(CH3)2SO] was incubated with P450 2S1 (total volume of 0.5 mL) at 37 °C for 15 min under anaerobic conditions. (A) 4-HONH-biphenyl in 100mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA was incubated at 37 °C for 15 min. (B) 4-HONH-biphenyl incubated with all P450system components, with the exception of P450 2S1. (C) 4-HONH-biphenyl incubated with all components of the P450 2S1 system, with theexception of the NADPH-generating system. (D) 4-HONH-biphenyl incubated with P450 2S1 in the presence of NPR and an NADPH-generatingsystem. (E) Synthetic standard of 4-HONH-biphenyl. The UV absorbance was integrated over the range of 200−400 nm.



= 4.5 min, m/z 170, [MH]+ (Figure 2A)], 4-HONH-biphenyl[tR = 5.5 min, m/z 186, [MH]+ (Figure 2B)], 4-NO-biphenyl[tR = 6.8 min, m/z 184 [MH]+ (Figure 2C)], andazoxybiphenyl [tR = 8.1 min, m/z 351 [MH]+ (Figure 2D)]were detected via LC−MS (APCI+) analyses of the incubationsamples. However, 4-NO-biphenyl and azoxybiphenyl were notdetected when LC−MS (electrospray ionization, ESI+) wasemployed.The decomposition of 2-HONH-naphthalene was more

rapid than that of 4-HONH-biphenyl. HPLC analyses of theincubation mixtures of 2-HONH-naphthalene with P450 2S1showed four major peaks (Figure S3 of the SupportingInformation), and minor peaks also appeared under someconditions. 2-NA (λmax = 275 nm), 2-HONH-naphthalene(λmax = 277 nm), 2-NO-naphthalene (λmax = 323 nm), andazoxynaphthalene (λmax = 346 nm) were eluted at retentiontimes of 3.7, 5.0, 6.4, and 7.9 min. 2-Nitronaphthalene (λmax =307 nm) eluted at a tR of 6.3 min under the same HPLCconditions (data not shown). 2-NA [tR = 3.7 min, m/z 144[MH]+ (Figure S4A of the Supporting Information)], 2-HONH-naphthalene [tR = 5.0 min, m/z 160 [MH]+ (FigureS4B of the Supporting Information)], 2-NO-naphthalene [tR =6.4 min, m/z 158 [MH]+ (Figure S4C of the SupportingInformation)], and azoxy naphthalene [tR = 7.9 min, m/z 299[MH]+ (Figure S4D of the Supporting Information)] weredetected via LC−MS (APCI+) analyses of the incubationsamples.Similarly, HPLC analyses of the anaerobic incubation

mixtures of P450 and 2-HONH-fluorene (which disappearedcompletely after incubations) also showed four major peaks(Figure S5 of the Supporting Information), suggesting extremeinstability under these conditions. 2-AF (λmax = 300 nm), 2-HONH-fluorene (λmax = 282 nm), 2-NO-fluorene (λmax = 372nm), and azoxyfluorene (λmax = 379 nm) were eluted at tRvalues of 4.4, 5.5, 6.8, and 8.3 min, respectively. 2-Nitrofluorene(λmax = 335 nm) appeared at a tR of 6.75 min under the sameHPLC conditions. 2-AF [tR = 4.4 min, m/z 182 [MH]+ (Figure

S6A of the Supporting Information)], 2-NHOH-fluorene [tR =5.5 min, m/z 198 [MH]+ (Figure S6B of the SupportingInformation)], 2-NO-fluorene [tR = 6.8 min, m/z 196 [MH]+

(Figure S6C of the Supporting Information)], and azoxy-fluorene [tR = 8.3 min, m/z 375 [MH]+ (Figure S6D of theSupporting Information)] were observed via LC−MS (APCI+)analyses of the incubation samples.

Anaerobic Reduction of HONH-5F 203, 4-HONH-Biphenyl, and 2-HONH-Fluorene with Other HumanP450s. HONH-5F 203 can be reduced by P450 2S1 to 5F 203under anaerobic conditions and (to a lesser extent) underaerobic conditions, as shown previously.53 Interestingly, when itwas incubated with P450 1A1 or 2W1 at 37 °C for 30 minunder anaerobic conditions (Figure S12 of the SupportingInformation), HONH-5F 203 (tR = 6.1 min) was also bereduced to 5F 203 (tR = 6.4 min). To evaluate the relativecatalytic efficiency of P450 2S1 for the reduction of aromatic N-hydroxylamines, anaerobic incubations of 4-HONH-biphenyl(Figure S13 of the Supporting Information) and 2-HONH-fluorene (Figure S14 of the Supporting Information) with P4501A1, 1A2, 2S1, 2W1, and 3A4 were conducted in parallel. 4-HONH-biphenyl (tR = 5.5 min) disappeared almostcompletely, and significantly larger amounts of azoxybiphenyl(tR = 8.1 min) were formed in the presence of P450s [exceptfor P450 2S1 (Figure S13A,B,D,E of the SupportingInformation)], suggesting higher degrees of disproportionation.In addition to a lower level of disproportionation, more of thereduction product 4-ABP [tR = 4.5 min (Figure S13C of theSupporting Information)] was generated with P450 2S1.Similar results were observed in the anaerobic reduction of 2-HONH-fluorene with various P450s (Figure S14 of theSupporting Information), where the largest amount of 2-AF[tR = 4.3 min (Figure S14C of the Supporting Information)]was produced with P450 2S1.

Reduction of 2-(3-Methyl-4-nitrosophenyl)-5-fluoro-benzothiazole (NO-5F 203) and 4-NO-Biphenyl byNADPH and P450 2S1. Some aryl nitroso compounds are

Figure 2. LC−MS spectra of incubation products of 4-HONH-biphenyl with P450 2S1: (A) 4-ABP (tR = 4.6 min), (B) 4-HONH-biphenyl (tR = 5.5min), (C) 4-NO-biphenyl (tR = 6.8 min), and (D) azoxybiphenyl (tR = 8.1 min).



known to be nonenzymatically reduced by NAD(P)H to N-hydroxyarylamines,64 which could be further reduced by P4502S1 to arylamines (Scheme 3). Freshly prepared NO-5F 20353

(containing trace amounts of 5F 203 and HONH-5F 203) wasincubated with P450 2S1 at 37 °C for 30 min under anaerobicconditions (Figure 3) and aerobic conditions (data not shown).The tR values of 5F 203 (λmax = 347 nm), HONH-5F 203 (λmax

= 338 nm), and NO-5F 203 (λmax = 363 nm) were 6.3, 6.0, and7.4 min, respectively, consistent with our previously reporteddata.53 In the absence of P450 2S1, HONH-5F 203 was formedvia reduction of NO-5F 203, as the most abundant peak(Figure 3B, in the presence of NPR and an NADPH-generatingsystem), whereas in the absence of an NADPH-generatingsystem, HONH-5F 203 was less abundant than 5F 203 (Figure3C), suggesting that NADPH was capable of nonenzymaticallyreducing NO-5F 203 to HONH-5F 203. When P450 2S1 wasadded, NO-5F 203 disappeared and the predominant productwas 5F 203 (the arylamine), indicating that P450 2S1 cancatalyze the reduction of HONH-5F 203 to 5F 203 (Figure3D). These results were in accord with our previousobservations.53 Freshly prepared 4-NO-biphenyl (containingtrace 4-ABP and azoxybiphenyl) was incubated with P450 2S1

Scheme 3. Reduction of Aromatic Nitroso and NitroCompounds by NADPH and P450 2S1

Figure 3. Anaerobic incubations of NO-5F 203 with P450 2S1. Freshly prepared NO-5F 203 [containing trace amounts of 5F 203 and HONH-5F203, 5 μL of an approximately 2 mM solution in (CH3)2SO] was incubated with P450 2S1 (total volume of 0.5 mL) at 37 °C for 30 min underanaerobic conditions. (A) NO-5F 203 in 100 mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA, incubated at 37 °C for 30 min. (B) NO-5F 203 incubated with all P450 system components, with the exception of P450 2S1. (C) NO-5F 203 incubated with all components of the P4502S1 system, with the exception of the NADPH-generating system. (D) NO-5F 203 incubated with P450 2S1 in the presence of NPR and anNADPH-generating system. (E) Synthetic standard of NO-5F 203. The UV absorbance was integrated over the range of 200−400 nm.



at 37 °C for 30 min under anaerobic (Figure 4) and aerobicconditions (data not shown). 4-ABP (λmax = 250 nm), 4-NHOH-biphenyl (λmax = 273 nm), 4-NO-biphenyl (λmax = 345nm), and azoxybiphenyl (λmax = 357 nm) were eluted at tRvalues of 4.5, 5.5, 6.8, and 8.2 min, respectively. The reductionof 4-NO-biphenyl occurred at an appreciable level only whenNPR and an NADPH-generating system were present (Figure4B,D), suggesting that the reduction of 4-NO-biphenyl isenzymatic. 4-NO-biphenyl disappeared completely with P4502S1 in the presence of NPR and an NADPH-generating system(Figure 4D). The ability of NPR to reduce 4-NO-biphenyl wasnot very obvious because of the high instability of 4-NHOH-biphenyl (rapid disproportionation) and the competitivecondensation reaction between 4-NHOH-biphenyl and 4-NO-biphenyl to form azoxybiphenyl. Nevertheless, it wasclear that a larger amount of 4-ABP was formed in the presenceof P450 2S1 (Figure 4C,D), indicating that P450 2S1 cancatalyze the reduction of NHOH-biphenyl to 4-ABP.

Reduction of NO2-IQ and 2-Nitronaphthalene byNADPH and P450 2S1. Some heterocyclic and aryl nitrocompounds could be reduced by P450 2S1, as exemplified byNO2-IQ and 2-NO2-naphthalene (Scheme 3). NO2-IQ wasincubated without (Figure 5A) or with (Figure 5B) P450 2S1in the presence of NPR and an NADPH-generating system at37 °C for 20 min under anaerobic conditions. In the absence ofP450 2S1, NO2-IQ was partially reduced to yield HONH-IQ[[M + H]+, m/z 215 (Figure S7A of the SupportingInformation)] as the only product; however, in the presenceof P450 2S1, NO2-IQ disappeared completely and IQ [[M +H]+, m/z 199 (Figure S7B of the Supporting Information)] wasproduced, in addition to HONH-IQ, indicating that P450 2S1can catalyze the reduction of HONH-IQ to IQ (the amine).Similar to the results observed for NO2-IQ, in the absence ofP450 2S1, 2-NO2-naphthalene was also partially reduced toyield 2-NHOH-naphthalene as the major product (Figure 5C).However, in the presence of P450 2S1, 2-NA was formedalmost exclusively and no 2-NHOH-naphthalene was present at

Figure 4. Anaerobic incubations of 4-NO-biphenyl with P450 2S1. Freshly prepared 4-NO-biphenyl [containing trace 4-ABP and azoxybiphenyl, 5μL of an approximately 2 mM solution in (CH3)2SO] was incubated with P450 2S1 (total volume of 0.5 mL) at 37 °C for 30 min under anaerobicconditions. (A) 4-NO-biphenyl in 100 mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA, incubated at 37 °C for 30 min. (B) 4-NO-biphenyl incubated with all P450 system components, with the exception of P450 2S1. (C) 4-NO-biphenyl incubated with all components of theP450 2S1 system, with the exception of the NADPH-generating system. (D) 4-NO-biphenyl incubated with P450 2S1 in the presence of NPR andan NADPH-generating system. (E) Synthetic standard of 4-NO-biphenyl. The UV absorbance was integrated over the range of 200−400 nm.



a discernible level (Figure 5D), suggesting that P450 2S1 cancatalyze the reduction of 2-NHOH-naphthalene as well.HONH-5F 203 Binding to P450 2S1. Titration of

HONH-5F 203 with P450 2S1 resulted in a concentration-dependent decrease in absorbance at 417 nm and an increase at458 nm (Figure 6), indicative of interactions between HONH-5F 203 and the heme iron.Steady-State Kinetic Data for Anaerobic Reduction of

HONH-5F 203 and Rates of Reduction of 4-NHOH-Biphenyl, 2-NHOH-Naphthalene, and 2-NHOH-Fluoreneby P450 2S1. Steady-state kinetic analysis of the reduction ofHONH-5F 203 with P450 2S1 yielded a rather linear rate−substrate relationship, showing little saturation with HONH-5F203 over the concentration range of 0−200 μM (Figure 7). kcatand Km values could not be determined by this method, but theslope provides an estimate of the catalytic efficiency (kcat/Km)of P450 2S1 [(5.5 ± 0.4) × 104 M−1 min−1, or ∼103 M−1 s−1].The rates of reduction of 4-NHOH-biphenyl (0.18 ± 0.01min−1), 2-NHOH-naphthalene (2.4 ± 0.8 min−1), and 2-NHOH-fluorene (0.34 ± 0.12 min−1) by P450 2S1 weremeasured at a single substrate concentration of 10 μM,considering only the amine formation contributed by P450 2S1catalysis (subtracting the amounts of amines in controlincubations in which P450 2S1 was absent).

■ DISCUSSIONIn this study, P450 2S1 did not exhibit oxidation activity withseveral common substrates for P450s or a number of aromaticand heterocyclic amines. Although some catalytic activity wasreported in the absence of NPR when oxygen surrogates were

Figure 5. Anaerobic incubations of NO2-IQ and 2-NO2-naphthalene with P450 2S1. NO2-IQ [5 μL of a 10 mM solution in (CH3)2SO] wasincubated with P450 2S1 (total volume of 0.5 mL) at 37 °C for 20 min under anaerobic conditions. (A) NO2-IQ incubated with all P450 systemcomponents, with the exception of P450 2S1. (B) NO2-IQ incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. (C)2-NO2-naphthalene incubated with all P450 system components, with the exception of P450 2S1. (D) 2-NO2-naphthalene incubated with P450 2S1in the presence of NPR and an NADPH-generating system. The UV absorbance was integrated over the range of 200−400 nm.

Figure 6. Binding spectra for titration of P450 2S1 with HONH-5F203. The calculated Kd was 30 ± 10 μM.



added,65,66 substrates for P450 2S1-catalyzed oxidation understandard conditions have not been identified.The reduction of N-hydroxylated compounds in rat and hog

liver extracts was noted several decades ago.67−70 For theenzymatic reduction, a microsomal (three-component enzymesystem containing cytochrome b5, NADH-cytochrome b5reductase, and an unidentified third component)67,68 and amitochondrial69,70 system have been proposed. In a study of thereduction of amidoxime prodrugs, stearoyl-CoA desaturase wasproposed to be involved in the microsomal reductions.71 Thethird component for the reduction of N-hydroxylamines in hogliver microsomes was isolated and characterized as a P450subfamily 2D enzyme.72 However, a human homologue has notbeen found. A novel molybdenum-containing enzyme from themitochondrial fraction, mitochondrial amidoxime reducingcomponent (mARC), was also suggested.73 Subsequentincubations with cytochrome b5, NADH cytochrome b5reductase, and mARC exhibited amidoxime reductase activityin vitro.74,75 The reduction of N-hydroxylamines in the absenceof the third component was also reported (i.e., onlycytochrome b5 and NADH cytochrome b5 reductase).76,77

One caveat of these latter studies is that the 4-HONH-biphenylused was not freshly prepared, and even in the presence ofascorbic acid, the disproportionation of 4-HONH-biphenyl(which also yields 4-ABP) was still significant under theexperimental conditions.Although most P450 reactions are oxidations,5 P450s can

also catalyze a wide range of reductions,1 as summarized byWisloki et al.78 and more recently by Testa.79 Generally in theP450 catalytic cycle, the ferrous P450 species binds O2 tightlyand oxidation occurs rapidly (after the addition of the secondelectron).1 However, in tissues where only a low concentrationof O2 is present, e.g., in hypoxic cancer cells,80 reductionbecomes more favorable. Ferrous P450 (Em,7 ∼ −300 mV)81,82

should be a reasonably good reducing agent if the substrate is inan appropriately bound form and has the potential to acceptelectrons. Alternatively, the binding of some substrates mayblock (or partially block) the incorporation of O2, which shouldallow the reduction to occur even under aerobic conditions.1 Itwas shown earlier that AQ4N (a di-N-oxide) can be reduced byboth P450 2W1 and 2S1 under anaerobic conditions.51

Subsequently, we found that 5F 203 can undergo P450 1A1-and 2W1-mediated bioactivation in the presence of O2, andHONH-5F 203 (one of the reactive intermediates) can bereduced by P450 2S1 under anaerobic conditions and, to a

lesser extent, aerobic conditions.53 In this report, we show thatP450 1A1 and 2W1 can also catalyze the reduction of HONH-5F 203 anaerobically, suggesting that the biotransformationpathways might be influenced by the O2 tension.The analgesic phenacetin is N-hydroxylated in rodents and

humans.54,55 Another metabolic pathway for phenacetin is O-deethylation, catalyzed by P450 1A2 to yield acetaminophen.83

Although acetaminophen is hepatotoxic at high doses,phenacetin induces a high incidence of tumors in the case ofchronic use of large doses to animals84 and was withdrawn fromthe market because of its possible carcinogenicity.85 Thebiotransformation pathways that lead to the formation of DNAadducts for the N-hydroxyphenacetin are similar to that for N-acetyl-2-AF (AAF) and many aromatic amines.26,55 We did notobserve any reduction of N-hydroxyphenacetin by P450 2S1,under any conditions used, suggesting that P450 2S1 probablyis not involved in the biotransformation of N-hydroxyphena-cetin. However, it should be pointed out that N-hydroxyphe-nacetin is a hydroxamic acid, not a hydroxylamine, and we didnot pursue the issue of whether P450 2S1 (or other P450s)reduces other hydroxamic acids.The results in this study demonstrate that human P450 2S1

is capable of catalyzing the reduction of several N-hydroxyaromatic amines and HAAs, especially under anaerobicconditions. P450 2S1 appeared to be the most efficient enzymeamong several P450s examined, at least for the anaerobicreductions of 4-HONH-biphenyl and 2-HONH-fluorene. It isworth noting that 4-HONH-biphenyl, 2-HONH-naphthalene,and 2-HONH-fluorene are very unstable molecules, even in theabsence of O2, and readily undergo disproportionationreactions to yield amine, nitroso, and azoxy derivatives. Thus,the formation of aromatic amines from N-hydroxylamines doesnot necessarily mean that reduction is occurring; i.e., thedifference between the amounts of amine formed with andwithout P450 2S1 reflects the enzymatic activity, and therefore,the rates of anaerobic reductions of 4-HONH-biphenyl, 2-HONH-naphthalene, and 2-HONH-fluorene were measured inthis way. The presence of NADPH slowed the disproportio-nation to various extents (Figure 1B, and Figures S3B and S5Bof the Supporting Information) when compared to theincubation controls (Figures 1A, 3A, and 5A), presumablybecause of nonenzymatic reduction of nitroso species. On thecontrary, P450 2S1 accelerated the process of disproportiona-tion (Figure 1C, and Figures S3C and S5C of the SupportingInformation), and some nonreductive pathways for the reactionof oxidized amines have been described previously.86

The nitroso derivatives of 5F 203 and 4-ABP were reducedby NADPH with NPR to N-hydroxylamines (Figure 7B) underanaerobic conditions. In the presence of P450 2S1, as well asNPR, NO-5F 203 was reduced to 5F 203 (amine) almostcompletely (Figure 3D). P450 2S1-catalyzed anaerobicreduction of 4-HONH-biphenyl occurred but was not asefficient as that with HONH-5F 203 (Figure 4D). A largeramount of azoxybiphenyl (Figure 4D) compared to theincubation control (Figure 4A) is due to the fact that nodisproportionation can occur because no 4-HONH-biphenyl isformed in the control reaction. Nitroso derivatives of aromaticamines and HAAs can react with hemoglobin-β Cys-93 in thebloodstream to form arylamine−hemoglobin sulfinamideadducts in experimental animals.87,88 4-ABP has been reportedto have a high fraction of dose (>5%) bound to hemoglobin assulfinamide adducts.89 Reduction of nitroso compounds maydecrease the level of formation of hemoglobin adducts. The

Figure 7. Catalysis of anaerobic reduction of HONH-5F 203 by P4502S1 as a function of substrate concentration.



nitroso derivative of IQ has been shown to be reduced by NPRto form the N-hydroxylamine and a trace amount of IQ.64

Similarly, the nitro derivatives of IQ and 2-NA are also reducedby NPR to N-hydroxylamines as major products (Figure 5A,C)under anaerobic conditions. If P450 2S1 is also present, N-hydroxylamines are reduced to yield IQ and 2-NA [amines(Figure 5B,D)].In conclusion, our results indicate that human P450 2S1 is

capable of catalyzing the reduction of N-hydroxylamine,nitroso, and nitro derivatives of at least several carcinogenicaromatic amines and HAAs to yield amines. This may representa detoxication pathway for aromatic amines and HAAs,although at this time we cannot judge the extent of the invivo contribution.

■ ASSOCIATED CONTENT*S Supporting InformationHPLC chromatograms of anaerobic incubations of HONH-phenacetin with P450 2S1, HPLC chromatograms of aerobicincubations of 4-HONH-biphenyl with P450 2S1, HPLCchromatograms of anaerobic incubations of 2-HONH-naph-thalene with P450 2S1, mass spectra of incubation products of2-HONH-naphthalene with P450 2S1, HPLC chromatogramsof anaerobic incubations of 2-HONH-fluorene with P450 2S1,mass spectra of incubation products of 2-HONH-fluorene withP450 2S1, LC−MS chromatograms of HONH-IQ and IQ, UVspectra of 5F 203, HONH-5F 203, and NO-5F 203, UV spectraof 4-ABP, 4-HONH-biphenyl, 4-NO-biphenyl, 4-NO2-biphen-yl, and azoxybiphenyl, UV spectra of 2-NA, 2-HONH-naphthalene, 2-NO-naphthalene, 2-NO2-naphthalene, andazoxynaphthalene, UV spectra of 2-AF, 2-HONH-fluorene, 2-NO-fluorene, 2-NO2-fluorene, and azoxyfluorene, HPLCchromatograms of anaerobic incubations of HONH-5F 203with P450 1A1 and 2W1, HPLC chromatograms of anaerobicincubations of 4-HONH-biphenyl with P450 1A1, 1A2, 2S1,2W1, and 3A4, and HPLC chromatograms of anaerobicincubations of 2-HONH-fluorene with P450 1A1, 1A2, 2S1,2W1, and 3A4. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Department of Biochemistry, Vanderbilt University School ofMedicine, 638 Robinson Research Building, 2200 Pierce Ave.,Nashville, TN 37232-0146. E-mail: [email protected]. Telephone: (615) 322-2261. Fax: (615) 322-4349.FundingThis work was supported in part by the U.S. Public HealthService (F.P.G., R37 CA090426, P30 ES000267).NotesThe authors declare no competing financial interest.

■ ABBREVIATIONS2-AAF, 2-acetylaminofluorene; 4-ABP, 4-aminobiphenyl; 2-AF,2-aminofluorene; APCI, atmospheric-pressure chemical ioniza-tion; AQ4, 1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dehy-droxyanthracene-9,10-dione; AQ4N, 1,4-bis{[2-(dimethylami-no-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione;ESI, electrospray ionization; 5F 203, 2-(4-amino-3-methyl-phenyl)-5-fluorobenzothiazole; HAAs, heterocyclic aromaticamines; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesul-fonic acid; HONH, hydroxylamino; HONH-5F 203, 2-(4-

hydroxylamino-3-methylphenyl)-5-fluorobenzothiazole; IQ, 2-amino-3-methylimidazo[4,5-f ]quinoline; LC−MS, liquid chro-matography−mass spectrometry; mARC, mitochondrial ami-doxime reducing component; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline; 2-NA, 2-naphthylamine;NAT, N-acetyltransferase; NO, nitroso; NO-5F 203, 2-(3-methyl-4-nitrosophenyl)-5-fluorobenzothiazole; NO2, nitro;NPR, NADPH-P450 reductase; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SULT, sulfotransferase.

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Reduction of Aromatic and Heterocyclic Aromatic N ...courses.washington.edu/medch582/PDFs/2014_Feb10_Robert.pdfP450-catalyzed N-hydroxylation is usually involved as the initial step