Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 ...

12 Pages·2013·2.19 MB·English

Article
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N‑
Reduction of Aromatic and Heterocyclic Aromatic Hydroxylamines
by Human Cytochrome P450 2S1
*
Kai Wang and F. Peter Guengerich
DepartmentofBiochemistryandCenterinMolecularToxicology,VanderbiltUniversity,SchoolofMedicine,638RobinsonResearch
Building, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, United States
*
S Supporting Information
ABSTRACT: Many aromatic amines and heterocyclic
aromatic amines (HAAs) are known carcinogens for animals,
and there is also strong evidence of some in human cancer.
Theactivationofthesecompounds,includingsomearylamine
drugs, involves N-hydroxylation, usually by cytochrome P450
enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We
previously demonstrated that the bioactivation product of the
anticanceragent2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole(5F203),anN-hydroxylamine,canbereducedbyP4502S1
toitsamineprecursorunderanaerobicconditionsand,toalesserextent,underaerobicconditions[Wang,K.,andGuengerich,F.
P.(2012)Chem.Res.Toxicol.25,1740−1751].Inthestudypresentedhere,wetestedthehypothesisthatP4502S1isinvolvedin
thereductive biotransformationofknowncarcinogenicaromaticaminesandHAAs.TheN-hydroxylaminesof4-aminobiphenyl
(4-ABP),2-naphthylamine(2-NA),and2-aminofluorene(2-AF)weresynthesizedandfoundtobereducedbyP4502S1under
both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic
conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded
amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines
testedwereoxidizedbyP4502S1.TheseresultssuggestthatP4502S1maybeinvolvedinthereductivedetoxicationofseveralof
the activated products of carcinogenic aromatic amines and HAAs.
■
INTRODUCTION detected in beer, wine,14 and cigarette smoke condensate.15,16
The Maillard reaction is believed to be involved in the
P450 enzymes catalyze a variety of reactions and are of
significant importance in the areas of drug metabolism and formation of the imidazo-containing HAAs when amino acids
toxicology.1,2Onthebasisofanalysesofmarketeddrugs,∼75% and carbohydrates are heated together,17,18 and other HAAs
of drug metabolism reactions involve P450s; five P450s (1A2, may form through the pyrolysis of amino acids and proteins.19
2C9, 2C19, 2D6, and 3A4) account for ∼90% of the P450 Extensive studies have shown that HAAs are highly genotoxic
reactions.3,4 Another recent analysis indicated that 66% of in bacterial and mammalian cells and exhibit strong
carcinogenic effects in experimental animals.20−23
carcinogens are bioactivated by P450s, and three Family 1
P450s show dominant roles, involved in ∼50% of the total The role of P450s in the bioactivation of aromatic amines
and HAAs has been well-documented24−26 and reviewed.7,13,27
activations attributed to P450s (1A1, 20%; 1A2, 17%; 1B1,
11%).5 P450-catalyzedN-hydroxylationisusuallyinvolvedastheinitial
step in the process of chemical carcinogenesis with such
Aromatic amines [arylamines (Scheme 1); e.g., 4-amino-
biphenyl (4-ABP), 2-naphthylamine (2-NA), 2-aminofluorene compounds (Scheme 2).26,28 The N-hydroxylamines can be
furtheractivatedtomorereactiveesters,byN-acetyltransferases
(2-AF)] are important industrial intermediates for the
(NAT), sulfotransferases (SULT), and other enzymes.7
production of azo dyes and complex chemicals and are also
usedasantioxidantsinrubbermanufacturingprocesses.6,7Rehn Subsequently, the resulting ester breaks down to yield an
firstreportedtheassociationofexposuretoaromaticaminesin extremely unstable intermediate, a nitrenium ion, which reacts
with DNA predominantly at position C8 of guanine bases and
1895withthe incidenceofbladdercanceramong Germanand
Swiss workers in aniline dye factories.8 Further studies have potentiallycausesDNAmutations(alternatively, thenitrenium
ioncanrearrange, andtheactive moleculewillreactwith aN2
shown that aromatic amines induce tumors at multiple sites in
guanineatom).29Family1P450enzymes(1A1,1A2,and1B1)
rodents, dogs, and other experimental laboratory animals as
well.6,7,9−11 are responsible for the metabolic activation of many aromatic
Heterocyclic aromatic amines [HAAs (Scheme 1); e.g., 2- amines, HAAs, and polycyclic aromatic hydrocarbons.24,30−36
amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8- TheexpressionofP450genesinthisfamilyisregulatedbythe
dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)] are produced Received: April8, 2013
in grilled and charred meats.12,13 Several HAAs have also been Published: May18, 2013
©2013AmericanChemicalSociety 993 dx.doi.org/10.1021/tx400139p|Chem.Res.Toxicol.2013,26,993−1004
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Scheme 1. Structures of Carcinogenic Aromatic and Heterocyclic Aromatic Amines
Scheme 2. Biotransformation Pathways of Aromatic and Heterocyclic Aromatic Amines
aryl hydrocarbon receptor (AhR).37 P450 1A2 is expressed phenacetin54) was not reduced by P450 2S1. Our results
almostexclusivelyinliverinhumans(10−15%oftotalP450),38 suggest that P450 2S1 may be involved in the detoxication of
but P450 1A1 and 1B1 are generally not present in liver but the activated products of a number of aromatic amines and
expressed in extrahepatic tissues at various levels.39 A number HAAs.
■
ofinvitroandinvivostudieshaveshownthatP4501A2playsa
majorroleinthebioactivationofaromaticaminesandHAAsin EXPERIMENTAL PROCEDURES
rodents and humans.30,40,41
Caution: Many of the compounds used in this study are carcinogens
The human genome has 57 P450 genes. P450 2S1 is one of
∼13 that are classified as “orphans” with only limited evidence and should be handled with care! Gloves, lab coats, and safety goggles
should be worn when handlingthese compounds.
offunction.42P4502S1mRNAhasbeendetectedinliver,skin,
tumors,andothertissues,43−46andthegeneisregulatedbythe Adilrlecctolmymweirtchioalulyt ofubrttahineerdpsuorlivfiecnattsioann.d4o-Nthietrropchheemneictoallse,w1e-raemuinseod-
AhR.47,48 The sites of expression and mode of regulation naphthalene,2-aminonaphthalene,and2-nitrofluorenewerepurchased
suggest that P450 2S1 might be involved in the process of from Sigma-Aldrich (St. Louis, MO). 2-Nitronaphthalene, 4-nitro-
chemical carcinogenesis.45 Recently, attenuation of P450 2S1 biphenyl,NO-IQ,NO-MeIQx,andNO-PhIPwerekindlyprovided
2 2 2
by siRNA has been shown to enhance cell proliferation and by Robert. J. Turesky (Wadsworth Center, Albany, NY). NMR data
migration in bronchial epithelial cells.49 However, a number of wereobtainedwitha400MHzBrukerNMRspectrometer,usingthe
carcinogenshavebeentestedandshownnottobeactivatedby solventsnoted,atVanderbilt.UVspectrawereobtained(online)with
P4502S1.50Retinoicacidwasreported asasubstrateforP450 a Waters Acquity UPLC system equipped with a photodiode array
2S1,44butsubsequenteffortsbyothershavefailedtoreproduce detector (Waters, Milford, MA).
theseresults.51,52Thefirstrepeatedlydemonstratedreactionfor waCshpermepicaarledSynutshinegsisa.Nm-Hoyddifirocxaytipohnenoafceatin.pTrehveiotuitslleycormeppoorutnedd
P4502S1istheanaerobicreductionofadi-N-oxideanticancer procedure.54 4-Nitrophenetole (0.10 g, 0.60 mmol) was reduced to
prodrug,1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8- N-hydroxyphenetidinein2mLofaCHOH/HOmixture(3:1,v/v)
2 5 2
dihydroxyanthracene-9,10-dione (AQ4N), to its amino deriv- byZndust(0.16 g,2.4mmol)inthepresenceof0.032gofNHCl.
4
ative, 1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dehydrox- Thereactionmixturewasdilutedwith4mLofcoldHOandextracted
2
yanthracene-9,10-dione (AQ4).51,52 Previously, we demonstra- with (CH)O (3 × 5 mL). The combined (CH)O extracts were
2 5 2 2 5 2
ted that an N-hydroxylarylamine, 2-(4-hydroxylamino-3-meth- evaporatedtodrynessinvacuoat23°C.Theresiduewasdissolvedin2
ylphenyl)-5-fluorobenzothiazole (HONH-5F 203), can be mL of (C2H5)2O in the presence of a slurry of NaHCO3 (0.08 g in
reduced to its amine precursor, 2-(4-amino-3-methylphenyl)- 0.25 mL of water) at 0 °C, and 2.5% acetyl chloride (47 mg, 0.60
5-fluorobenzothiazole (5F 203, an anticancer agent), by P450 mmol) in (C2H5)2O (w/v) was added slowly to the mixture. The
2S1 under anaerobic conditions.53 resultingmixturewasstirredfor2handextractedwith(C2H5)2O(3×
5mL).Thecombined(CH)Olayerswerewashedsequentiallywith
In this study, several N-hydroxylarylamines and nitroso and 2 5 2
HO and brine and dried over anhydrous NaSO. The solvent was
nitroderivatives ofcommon carcinogenicaromatic aminesand rem2 ovedinvacuo,andflashcolumnchromatogr2aphy4 onsilicagelusing
HAAsweresynthesizedandshowntobereducedbyP4502S1. (CH)OyieldedN-hydroxyphenacetinascolorlesscrystals(0.053g,
2 5 2
TheratesofP4502S1-catalyzedanaerobicreductionsoftheN- 45%yieldfrom4-nitrophenetole,mp101−103°C,lit.55mp103°C):
hydroxylarylaminesweredetermined.However,N-hydroxyphe- 1H NMR (400 MHz, CDCl) δ 7.37 (d, J = 8.8 Hz, aromatic, 2H),
3
nacetin (the activated product of the analgesic drug 6.85(d,J=8.9Hz,aromatic,2H),4.01(q,J=7.0Hz,2H,CHCH-),
3 2
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2.16(s,3H,CHCO−),1.40(t,J=7.0Hz,3H,CHCH−);GC−MS NADPH-generatingsystemandbufferconditionswerethesameasin
3 3 2
(electron impact)m/z 179, 137, 108. atypicalincubation,videsupra.Astandardsolution(5μL)wasadded
N-HydroxylaminesandNitrosoCompounds.HONH-5F203,534- toeachenzymereactionmixture(volumeof500μL)andeachmixture
HONH-biphenyl, 2-HONH-naphthalene, 2-HONH-fluorene, and incubatedat37°C.ReactionswereterminatedbyadditionofCHCl
2 2
HONH-PhIP were prepared by the reduction of the corresponding (1.0 mL) after 0, 1, 2, 3, 4, and 5 min. For each compound,
nitro derivatives with hydrazine and Pd/C at 0 °C for 30 min to 2 incubations were performed in duplicate at every time point.
h.56,57HONH-IQandHONH-MeIQxwerepreparedbythereduction Incubations devoid of P450 2S1 were performed as controls. 1-
of nitro analogues with ascorbic acid in (CH)SO as described by Naphthylamine (5 μL, 500 μM) was added to each sample as an
3 2
Tureskyetal.57NO-5F203and4-NO-biphenylwerepreparedbythe internal standard, and the mixtures were centrifuged. The isolated
oxidationofHONH-5F203and4-HONH-biphenylwithKFe(CN) organiclayersweretransferredanddriedunderastreamofN,andthe
3 6 2
underambient conditions for 3h asreported previously.53 residues were dissolved inCHCN for HPLC analysis.
3
Enzyme Preparations. Recombinant human P450 2S1 was HPLCandLC−MSAnalysisoftheReductionReactions.The
expressed and purified in Escherichia coli membranes as previously reduced reaction products of hydroxylamine, nitroso, and nitro
described.50 NADPH-P450 reductase (NPR) was expressed in E. coli compounds were analyzed using a Waters Acquity UPLC system
and purified asdescribed previously.58 equipped with a photodiode array detector (Waters) and an
IncubationConditions.Atypicalincubation59mixturecontained octadecylsilane (C18) reversed-phase column (5 μm, 2.1 mm × 250
purified P450 2S1 (0.5 μM), NPR (1 μM), 160 μM L-α-dilauroyl-sn- mm, Hypersil GOLD, Thermo Scientific, Odessa, TX). Absorbance
glycero-3-phosphocholine, an NADPH-generating system (10 mM was integrated (210−400 nm). The column temperature was
glucose6-phosphate,0.5mMNADP+,and4μg/mLyeastglucose-6- maintained at 25 °C. LC conditions for phenacetin, 4-ABP, 2-NA,
phosphatedehydrogenase),andtheappropriatesubstrate(100μM)in 2-AF,and5F203derivativeswereasdescribedpreviously.53SolventA
0.10 M sodium N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [5:95CH3CN/H2Omixturewith0.5%HCO2H(v/v/v)]andsolvent
(HEPES)buffer(pH7.4,containing1mMEDTA).Theincubations B[95:5 CH3CN/H2Omixturewith 0.5%HCO2H(v/v)]were used.
were performed at 37 °C for 30 min. Cold CHCl (2-fold volume) The gradient started from 5% B (v/v) during the first 2 min (0−2
wasaddedtoterminateeachreaction,andtheres2ult2ingmixtureswere min),increasedlinearlyto100%Bover5min(2−7min),washeldfor
separatedbycentrifugationat2000gfor3min.Thelowerorganiclayer 4.5min(7−11.5min),andthenreturnedto5%B(v/v)over0.5min
was transferred and dried under a N stream. The residue was (11.5−12min)beforere-equilibration(12−15min),ataflowrateof
2
dissolved in CHCNfor HPLC andLC−MS analysis. 0.5 mL/min.LCconditions fortheIQ,MeIQx,andPhIP derivatives
3
Anaerobic Incubations. Qualitative anaerobic experiments were were as follows. Solvent A contained 20 mM NH4CH3CO2 and 5%
performedinThunbergtubes,aspreviouslydescribed.53Atypical500 CH3CN (v/v), and solvent B contained NH4CH3CO2 (20 mM, pH
μLincubationmixturecontained0.5μMP450,1.0μMNPR,160μM 6.8) and 95% CH3CN (v/v). A linear gradient was employed with a
L-α-1,2-dilauoryl-sn-glycero-3-phosphocholine, 100 mM sodium flow rate of 0.5 mL/min, starting from 5% solvent B and reaching
HEPES buffer (pH 7.4), 1 mM EDTA, and 100 μM substrate [from 100%solventBat12min,andthepercentageofsolventBreturnedto
afreshlyprepared10mM(CH)SOstock].Whenthedesiredlevelof 5% (v/v) over 3min (12−15 min).
anaerobicity was achieved (aft3er2five cycles of vacuum and Ar), an LC−MS and LC−MS/MS analyses were performed on a Waters
NADPH-generatingsystemwasaddedtotheincubationmixturefrom Acquity UPLC system coupled to a Thermo-Finnigan LTQ ion trap
theneckofthetube.Thereactionmixtureswere incubatedat 37°C mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The
for10−30min,thereactionsterminatedbytheadditionof1.0mLof same LC conditions were used for each sample as in the HPLC
analysis.Samplesweregenerallyanalyzedintheatmospheric-pressure
coldCHCN, and the mixtures analyzed by HPLC.
3 chemical ionization (APCI) positive ion mode. MS conditions were
Forcomparisons,anaerobicincubationswereconductedwithP450
1A1,1A2,2S1,2W1,and3A4atthesameconcentration(0.2μM)for optimized foreachaminederivative asfollows:capillary temperature,
275°C;vaporizertemperature,450°C;sourcevoltage,6.0kV;sheath
10min.
Steady-StateKineticMeasurementsofAnaerobicReduction gasflow,50.0;auxiliarygasflow,5.0;sweepgasflow,5.0;andin-source
ofHONH-5F203.AnaerobicincubationswereconductedunderaN fragmentation, 10 V.
atmosphere in a glovebox (Labconco, Kansas City, MO). The2 Colorimetric Assay for N-Hydroxyarylamines.60 From a
concentrationsofP450andtheNADPH-generatingsystemandbuffer typical solution containing 5−10 mM N-hydroxyarylamine, a 1−2
conditions remained the same as those employed in the qualitative μL aliquot was added to a tube containing 0.96 mL of the color
studies.AstocksolutionwasmadeoffreshlypreparedHONH-5F203 reagent.60 The sample was mixed vigorously using a vortex device.
icnally(C.60HA3)2sSeOrie,saonfdsttahnedacrodnscoelnuttriaotniosnatwdaisffedreentetrmcoinnceedntcroatloiornimsewtrais- Amfitxetrur3e.mTinhe, 4s0amμpLleowf satsabmiliixzeedr (thHo3rPoOug4,hl2y0, amnMd )A5w35aswaadsdmedeatsourtehde,
preparedfromthestocksolutionbydilution.Reactionswithdifferent with reference to a sample containing all of the reagents except N-
substrateconcentrations(rangingfrom0to200μM)wereconducted, hydroxyarylamine as acontrol (ε535 =39200 M−1 cm−1).
in duplicate, at 37 °C for 10 min. After the reactions had been P4502S1BindingAssay(withHONH-5F203).Bindingspectra
quenchedwithCHCl (2-foldvolume),2-phenylbenzothiazole(5μL, of P450 with HONH-5F 203 were recorded with purified P450 2S1
2 2 (23 °C) using a UV−visible scanning spectrophotometer (Aminco
0.1mM)wasaddedtoeachreactionmixtureasaninternalstandard.
DW-2a, OLIS, Bogart, GA). A baseline was recorded (350−700 nm)
The mixtures were centrifuged at 2000g for 3 min, and the organic
usingtwo1.0 mLglasscuvettes (1cmpathlength)containing equal
layerswere transferred anddried underastreamof N.Theresidues
2 volumesof100mMpotassiumphosphatebuffer(pH7.4)containing
were dissolved in CHCN for HPLC analysis.
3 20% glycerol (v/v). Purified P450 2S1 was added to the sample
DeterminationofRatesofAnaerobicReductionof4-NHOH-
Biphenyl,2-NHOH-Naphthalene,and2-NHOH-Fluorenetothe cuvette(finalconcentrationof5μM).Thesamevolumeofthebuffer
CorrespondingAminesbyP4502S1.Anaerobicincubationswere wasaddedtothereferencecuvette.Equalamountsoffreshlyprepared
kcoinnedtuicctsetduduinesdefroraHNO2aNtmHo-5spFh2er0e3inreadugclotivoenb.oTx,haesrinatethseosfteraeddyu-csttiaotne abloiqthuoctusvoetfteHs.ODNiffHe-r5eFnc2e0s3pe[cdtirsasowlveerde cinoll(eCctHed3)2(S3O50]−w7e0r0enamdd)eadftteor
weremeasuredatasinglesubstrateconcentration(10μM).Standard ■anequilibrationperiodof1min[totalvolumeof(CH3)2SOof≤2%].
solutions (1 mM, determined by colorimetric assay) of 4-NHOH-
biphenyl, 2-NHOH-naphthalene, and 2-NHOH-fluorene in CHCN RESULTS
3
were prepared from freshly prepared samples. Under the conditions
Search for Oxidations of Potential Substrates with
used in this study, optimal P450 2S1 concentrations for 4-NHOH-
biphenyl,2-NHOH-naphthalene,and2-NHOH-fluorenewere500,25, P450 2S1. No detectable products were observed following
and 50 nM, respectively (the corresponding concentrations of NPR the incubation of aniline, 7-ethoxycoumarin, IQ, MeIQx, or
were 1, 0.5, and 0.5 μM, respectively). The concentrations of the PhIP with human P450 2S1, in typical aerobic incubation
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Figure 1. Anaerobic incubations of 4-HONH-biphenyl with P450 2S1. Freshly prepared 4-HONH-biphenyl [5 μL of a 10 mM solution in
(CH)SO]wasincubatedwithP4502S1(totalvolumeof0.5mL)at37°Cfor15minunderanaerobicconditions.(A)4-HONH-biphenylin100
3 2
mMsodiumHEPESbuffer(pH7.4)containing1mMEDTAwasincubatedat37°Cfor15min.(B)4-HONH-biphenylincubatedwithallP450
system components, with the exception of P450 2S1. (C) 4-HONH-biphenyl incubated with all components of the P450 2S1 system, with the
exceptionoftheNADPH-generatingsystem.(D)4-HONH-biphenylincubatedwithP4502S1inthepresenceofNPRandanNADPH-generating
system.(E) Synthetic standard of 4-HONH-biphenyl. TheUV absorbance was integrated over the range of 200−400 nm.
systems containing NPR and NADPH, as judged by HPLC− incubations of all three N-arylhydroxylamines with P450 2S1
UV assays. No products were detected from an incubation (in the presence of NPR and an NADPH-generating system),
mixture of [14C]lauric acid with P450 2S1 using HPLC significantly larger amounts of the corresponding arylamines
(coupled online with scintillation counting). were formed [than under other conditions when P450 2S1 or
Anaerobic Incubation of N-Hydroxyphenacetin with an NADPH-generating system was absent (Figure 1, and
P450 2S1. N-Hydroxyphenacetin was incubated with P450 Figures S3 and S5 of the Supporting Information)], indicating
2S1at37°Cfor20minunderanaerobicoraerobicconditions, thatP4502S1reducedtheN-arylhydroxylaminestoarylamines
with an HPLC peak at a t of 4.7 min corresponding to N- (Scheme2).Underaerobicincubationconditions(FigureS2of
R
hydroxyphenacetin. N-Hydroxyphenacetin was stable under all the Supporting Information), competitive disproportionations
conditions tested in this study (Figure S1 of the Supporting were favored that also yielded arylamines as products. The
Information). Neither P450 2S1, NPR, NADPH, nor the reduction of N-arylhydroxylamines by P450 2S1 was less
combination changed the N-hydroxyphenacetin to any apparent compared to what occurred under anaerobic
products. conditions.
Anaerobic Reduction of 4-HONH-Biphenyl, 2-HONH- HPLCanalysesoftheincubationof4-HONH-biphenylwith
Naphthalene, and 2-HONH-Fluorene with P450 2S1. N- P450 2S1 showed four major peaks (Figure 1). 4-ABP (λ =
max
Arylhydroxylamines are unstable substances and readily under- 250 nm),4-HONH-biphenyl (λ =273 nm), 4-NO-biphenyl
max
godisproportionationreactions(Scheme2)toyieldarylamines (λ = 345 nm), and azoxybiphenyl (λ = 357 nm) were
max max
and nitroso derivatives in neutral or basic media.61,62 The eluted at t values of 4.5, 5.5, 6.8, and 8.2 min, respectively. 4-
R
nitroso products can further condense with N-arylhydroxyl- Nitrobiphenyl (λ = 310 nm) appeared at a t of 6.6 min
max R
amines to produce azoxy compounds.63 In anaerobic underthesameHPLCconditions(datanotshown).4-ABP[t
R
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Figure2.LC−MSspectraofincubationproductsof4-HONH-biphenylwithP4502S1:(A)4-ABP(t =4.6min),(B)4-HONH-biphenyl(t =5.5
R R
min), (C)4-NO-biphenyl (t = 6.8 min), and(D) azoxybiphenyl (t = 8.1 min).
R R
= 4.5 min, m/z 170, [MH]+ (Figure 2A)], 4-HONH-biphenyl S6A ofthe Supporting Information)], 2-NHOH-fluorene [t =
R
[t = 5.5 min, m/z 186, [MH]+ (Figure 2B)], 4-NO-biphenyl 5.5 min, m/z 198 [MH]+ (Figure S6B of the Supporting
R
[t = 6.8 min, m/z 184 [MH]+ (Figure 2C)], and Information)], 2-NO-fluorene [t = 6.8 min, m/z 196 [MH]+
R R
azoxybiphenyl [t = 8.1 min, m/z 351 [MH]+ (Figure 2D)] (Figure S6C of the Supporting Information)], and azoxy-
R
were detected via LC−MS (APCI+) analyses of the incubation fluorene [t = 8.3 min, m/z 375 [MH]+ (Figure S6D of the
R
samples.However,4-NO-biphenylandazoxybiphenylwerenot Supporting Information)] wereobservedviaLC−MS(APCI+)
detected when LC−MS (electrospray ionization, ESI+) was analyses of the incubation samples.
employed. Anaerobic Reduction of HONH-5F 203, 4-HONH-
The decomposition of 2-HONH-naphthalene was more Biphenyl, and 2-HONH-Fluorene with Other Human
rapid than that of 4-HONH-biphenyl. HPLC analyses of the P450s.HONH-5F203canbereducedbyP4502S1to5F203
incubation mixtures of 2-HONH-naphthalene with P450 2S1 under anaerobic conditions and (to a lesser extent) under
showed four major peaks (Figure S3 of the Supporting aerobicconditions,asshownpreviously.53Interestingly,whenit
Information), and minor peaks also appeared under some was incubated with P450 1A1 or 2W1 at 37 °C for 30 min
conditions. 2-NA (λ = 275 nm), 2-HONH-naphthalene under anaerobic conditions (Figure S12 of the Supporting
max
(λ = 277 nm), 2-NO-naphthalene (λ = 323 nm), and Information), HONH-5F 203 (t = 6.1 min) was also be
max max R
azoxynaphthalene (λ = 346 nm) were eluted at retention reduced to 5F 203 (t = 6.4 min). To evaluate the relative
max R
times of 3.7, 5.0, 6.4, and 7.9 min. 2-Nitronaphthalene (λ = catalyticefficiencyofP4502S1forthereductionofaromaticN-
max
307 nm) eluted at a t of 6.3 min under the same HPLC hydroxylamines, anaerobic incubations of 4-HONH-biphenyl
R
conditions (data not shown). 2-NA [t = 3.7 min, m/z 144 (Figure S13 of the Supporting Information) and 2-HONH-
R
[MH]+ (Figure S4A of the Supporting Information)], 2- fluorene(FigureS14oftheSupportingInformation)withP450
HONH-naphthalene [t = 5.0 min, m/z 160 [MH]+ (Figure 1A1, 1A2, 2S1, 2W1, and 3A4 were conducted in parallel. 4-
R
S4B ofthe Supporting Information)], 2-NO-naphthalene [t = HONH-biphenyl (t = 5.5 min) disappeared almost
R R
6.4 min, m/z 158 [MH]+ (Figure S4C of the Supporting completely, and significantly larger amounts of azoxybiphenyl
Information)], and azoxy naphthalene [t = 7.9 min, m/z 299 (t = 8.1 min) were formed in the presence of P450s [except
R R
[MH]+ (Figure S4D of the Supporting Information)] were for P450 2S1 (Figure S13A,B,D,E of the Supporting
detected via LC−MS (APCI+) analyses of the incubation Information)],suggestinghigherdegreesofdisproportionation.
samples. In addition to a lower level of disproportionation, more of the
Similarly, HPLC analyses of the anaerobic incubation reduction product 4-ABP [t = 4.5 min (Figure S13C of the
R
mixtures of P450 and 2-HONH-fluorene (which disappeared Supporting Information)] was generated with P450 2S1.
completely after incubations) also showed four major peaks Similar results were observed in the anaerobic reduction of 2-
(FigureS5oftheSupportingInformation),suggestingextreme HONH-fluorene with various P450s (Figure S14 of the
instability under these conditions. 2-AF (λ = 300 nm), 2- Supporting Information), where the largest amount of 2-AF
max
HONH-fluorene (λ = 282 nm), 2-NO-fluorene (λ = 372 [t = 4.3 min (Figure S14C of the Supporting Information)]
max max R
nm), and azoxyfluorene (λ = 379 nm) were eluted at t was produced with P450 2S1.
max R
valuesof4.4,5.5,6.8,and8.3min,respectively.2-Nitrofluorene Reduction of 2-(3-Methyl-4-nitrosophenyl)-5-fluoro-
(λ = 335 nm) appeared at a t of 6.75 min under the same benzothiazole (NO-5F 203) and 4-NO-Biphenyl by
max R
HPLCconditions.2-AF[t =4.4min,m/z182[MH]+(Figure NADPH and P450 2S1. Some aryl nitroso compounds are
R
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known to be nonenzymatically reduced by NAD(P)H to N- (containingtraceamountsof5F203andHONH-5F203)was
hydroxyarylamines,64 which could be further reduced by P450 incubated with P450 2S1 at 37°Cfor 30min underanaerobic
2S1 to arylamines (Scheme 3). Freshly prepared NO-5F 20353 conditions(Figure3)andaerobicconditions(datanotshown).
Thet valuesof5F203(λ =347nm),HONH-5F203(λ
R max max
Scheme 3. Reduction of Aromatic Nitroso and Nitro =338nm),andNO-5F203(λ =363nm)were6.3,6.0,and
max
Compounds by NADPH and P450 2S1 7.4 min, respectively, consistent with our previously reported
data.53IntheabsenceofP4502S1,HONH-5F203wasformed
via reduction of NO-5F 203, as the most abundant peak
(Figure3B,inthepresenceofNPRandanNADPH-generating
system), whereas in the absence of an NADPH-generating
system,HONH-5F203waslessabundantthan5F203(Figure
3C), suggesting thatNADPHwascapable ofnonenzymatically
reducing NO-5F 203 to HONH-5F 203. When P450 2S1 was
added, NO-5F 203 disappeared and the predominant product
was 5F 203 (the arylamine), indicating that P450 2S1 can
catalyze the reduction of HONH-5F 203 to 5F 203 (Figure
3D). These results were in accord with our previous
observations.53 Freshly prepared 4-NO-biphenyl (containing
trace 4-ABP and azoxybiphenyl) was incubated with P450 2S1
Figure3.AnaerobicincubationsofNO-5F203withP4502S1.FreshlypreparedNO-5F203[containingtraceamountsof5F203andHONH-5F
203,5μLofanapproximately 2mMsolutionin(CH)SO]wasincubatedwithP4502S1(totalvolumeof0.5mL)at37°Cfor30minunder
3 2
anaerobicconditions.(A)NO-5F203in100mMsodiumHEPESbuffer(pH7.4)containing1mMEDTA,incubatedat37°Cfor30min.(B)NO-
5F203incubatedwithallP450systemcomponents,withtheexceptionofP4502S1.(C)NO-5F203incubatedwithallcomponentsoftheP450
2S1 system, with the exception of the NADPH-generating system. (D) NO-5F 203 incubated with P450 2S1 in the presence of NPR and an
NADPH-generating system. (E) Synthetic standard of NO-5F203. The UVabsorbance was integrated over the range of 200−400 nm.
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Figure4.Anaerobicincubationsof4-NO-biphenylwithP4502S1.Freshlyprepared4-NO-biphenyl[containingtrace4-ABPandazoxybiphenyl,5
μLofanapproximately2mMsolutionin(CH)SO]wasincubatedwithP4502S1(totalvolumeof0.5mL)at37°Cfor30minunderanaerobic
3 2
conditions.(A)4-NO-biphenylin100mMsodiumHEPESbuffer(pH7.4)containing1mMEDTA,incubatedat37°Cfor30min.(B)4-NO-
biphenylincubatedwithallP450systemcomponents,withtheexceptionofP4502S1.(C)4-NO-biphenylincubatedwithallcomponentsofthe
P4502S1system,withtheexceptionoftheNADPH-generatingsystem.(D)4-NO-biphenylincubatedwithP4502S1inthepresenceofNPRand
anNADPH-generating system.(E) Syntheticstandard of 4-NO-biphenyl. TheUV absorbance was integrated over the range of 200−400 nm.
at 37 °C for 30 min under anaerobic (Figure 4) and aerobic Reduction of NO -IQ and 2-Nitronaphthalene by
2
conditions (data not shown). 4-ABP (λ = 250 nm), 4- NADPH and P450 2S1. Some heterocyclic and aryl nitro
max
NHOH-biphenyl(λ =273nm),4-NO-biphenyl(λ =345 compounds could be reduced by P450 2S1, as exemplified by
max max
nm), and azoxybiphenyl (λmax = 357 nm) were eluted at tR NO2-IQ and 2-NO2-naphthalene (Scheme 3). NO2-IQ was
values of 4.5, 5.5, 6.8, and 8.2 min, respectively. The reduction incubated without (Figure 5A) or with (Figure 5B) P450 2S1
in the presence of NPR and an NADPH-generating system at
of 4-NO-biphenyl occurred at an appreciable level only when
37°Cfor20minunderanaerobicconditions.Intheabsenceof
NPR and an NADPH-generating system were present (Figure
P450 2S1, NO -IQ was partially reduced to yield HONH-IQ
4B,D), suggesting that the reduction of 4-NO-biphenyl is 2
[[M + H]+, m/z 215 (Figure S7A of the Supporting
enzymatic. 4-NO-biphenyl disappeared completely with P450
Information)] as the only product; however, in the presence
2S1inthepresenceofNPRandanNADPH-generatingsystem
of P450 2S1, NO -IQ disappeared completely and IQ [[M +
(Figure4D).TheabilityofNPRtoreduce4-NO-biphenylwas 2
H]+,m/z199(FigureS7BoftheSupportingInformation)]was
not very obvious because of the high instability of 4-NHOH-
produced, in addition to HONH-IQ, indicating that P450 2S1
biphenyl (rapid disproportionation) and the competitive
can catalyze the reduction of HONH-IQ to IQ (the amine).
condensation reaction between 4-NHOH-biphenyl and 4- Similar to the results observed for NO -IQ, in the absence of
2
NO-biphenyl to form azoxybiphenyl. Nevertheless, it was P450 2S1, 2-NO -naphthalene was also partially reduced to
2
clearthatalargeramountof4-ABPwasformedinthepresence yield2-NHOH-naphthaleneasthemajorproduct(Figure5C).
of P450 2S1 (Figure 4C,D), indicating that P450 2S1 can However, in the presence of P450 2S1, 2-NA was formed
catalyze the reduction of NHOH-biphenyl to 4-ABP. almostexclusivelyandno2-NHOH-naphthalenewaspresentat
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Chemical Research in Toxicology Article
Figure 5. Anaerobic incubations of NO-IQ and 2-NO-naphthalene with P450 2S1. NO-IQ [5 μL of a 10 mM solution in (CH)SO] was
2 2 2 3 2
incubatedwithP4502S1(totalvolumeof0.5mL)at37°Cfor20minunderanaerobicconditions.(A)NO-IQincubatedwithallP450system
2
components,withtheexceptionofP4502S1.(B)NO-IQincubatedwithP4502S1inthepresenceofNPRandanNADPH-generatingsystem.(C)
2
2-NO-naphthaleneincubatedwithallP450systemcomponents,withtheexceptionofP4502S1.(D)2-NO-naphthaleneincubatedwithP4502S1
2 2
inthe presence ofNPR and anNADPH-generating system. TheUV absorbance was integrated over the range of 200−400 nm.
a discernible level (Figure 5D), suggesting that P450 2S1 can
catalyze 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-
dependentdecreaseinabsorbanceat417nmandanincreaseat
458nm(Figure6),indicative ofinteractionsbetweenHONH-
5F 203 and the heme iron.
Steady-StateKineticDataforAnaerobicReductionof
HONH-5F 203 and Rates of Reduction of 4-NHOH-
Biphenyl, 2-NHOH-Naphthalene, and 2-NHOH-Fluorene
by P450 2S1. Steady-state kinetic analysis of the reduction of
HONH-5F 203 with P450 2S1 yielded a rather linear rate−
substraterelationship,showinglittlesaturationwithHONH-5F
203overtheconcentrationrangeof0−200μM(Figure7).k
cat
andK valuescouldnotbedeterminedbythismethod,butthe
m
slope provides an estimate of the catalytic efficiency (k /K )
cat m
ofP4502S1[(5.5±0.4)×104M−1min−1,or∼103M−1s−1]. Figure 6. Binding spectra for titration of P450 2S1 with HONH-5F
The rates of reduction of 4-NHOH-biphenyl (0.18 ± 0.01 ■203. The calculated Kd was 30 ±10 μM.
min−1), 2-NHOH-naphthalene (2.4 ± 0.8 min−1), and 2-
NHOH-fluorene (0.34 ± 0.12 min−1) by P450 2S1 were DISCUSSION
measured at a single substrate concentration of 10 μM, In this study, P450 2S1 did not exhibit oxidation activity with
consideringonlytheamineformationcontributedbyP4502S1 several common substrates for P450s or a number of aromatic
catalysis (subtracting the amounts of amines in control and heterocyclic amines. Although some catalytic activity was
incubations in which P450 2S1 was absent). reported in the absence of NPR when oxygen surrogates were
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Chemical Research in Toxicology Article
lesser extent, aerobicconditions.53Inthisreport, weshow that
P4501A1and2W1canalsocatalyzethereductionofHONH-
5F 203 anaerobically, suggesting that the biotransformation
pathways might be influenced by the O tension.
2
The analgesic phenacetin is N-hydroxylated in rodents and
humans.54,55 Another metabolic pathway for phenacetin is O-
deethylation, catalyzed byP450 1A2toyield acetaminophen.83
Although acetaminophen is hepatotoxic at high doses,
phenacetin induces a high incidence of tumors in the case of
chronicuseoflargedosestoanimals84andwaswithdrawnfrom
the market because of its possible carcinogenicity.85 The
biotransformationpathwaysthatleadtotheformationofDNA
adducts for the N-hydroxyphenacetin are similar to that for N-
acetyl-2-AF(AAF)andmanyaromaticamines.26,55Wedidnot
observe any reduction of N-hydroxyphenacetin by P450 2S1,
Figure7.CatalysisofanaerobicreductionofHONH-5F203byP450 under any conditions used, suggesting that P450 2S1 probably
2S1as a function of substrate concentration. is not involved in the biotransformation of N-hydroxyphena-
cetin. However, it should be pointed out that N-hydroxyphe-
added,65,66 substrates for P450 2S1-catalyzed oxidation under nacetin is a hydroxamic acid, not a hydroxylamine, and we did
standard conditions have not been identified. not pursue the issue of whether P450 2S1 (or other P450s)
Thereduction ofN-hydroxylated compoundsinratandhog reduces other hydroxamic acids.
liver extracts was noted several decades ago.67−70 For the The results in this study demonstrate that human P450 2S1
enzymatic reduction, a microsomal (three-component enzyme is capable of catalyzing the reduction of several N-hydroxy
system containing cytochrome b , NADH-cytochrome b aromatic amines and HAAs, especially under anaerobic
reductase, and an unidentified thir5d component)67,68 and a5 conditions.P4502S1appearedtobethemostefficientenzyme
mitochondrial69,70systemhavebeenproposed.Inastudyofthe among several P450s examined, at least for the anaerobic
reductionofamidoximeprodrugs,stearoyl-CoAdesaturasewas reductions of 4-HONH-biphenyl and 2-HONH-fluorene. It is
proposed to be involved in the microsomal reductions.71 The worth noting that 4-HONH-biphenyl, 2-HONH-naphthalene,
thirdcomponentforthereductionofN-hydroxylaminesinhog and2-HONH-fluoreneareveryunstablemolecules,eveninthe
liver microsomes was isolated and characterized as a P450 absence of O2, and readily undergo disproportionation
subfamily2Denzyme.72However,ahumanhomologuehasnot reactions to yield amine, nitroso, and azoxy derivatives. Thus,
beenfound.Anovelmolybdenum-containingenzymefromthe theformationofaromaticaminesfromN-hydroxylaminesdoes
mitochondrial fraction, mitochondrial amidoxime reducing not necessarily mean that reduction is occurring; i.e., the
component (mARC), was also suggested.73 Subsequent difference between the amounts of amine formed with and
incubations with cytochrome b , NADH cytochrome b withoutP4502S1reflectstheenzymaticactivity,andtherefore,
5 5
reductase, and mARC exhibited amidoxime reductase activity the rates of anaerobic reductions of 4-HONH-biphenyl, 2-
invitro.74,75ThereductionofN-hydroxylaminesintheabsence HONH-naphthalene,and2-HONH-fluoreneweremeasuredin
of the third component was also reported (i.e., only this way. The presence of NADPH slowed the disproportio-
cytochrome b and NADH cytochrome b reductase).76,77 nationtovariousextents(Figure1B,andFiguresS3BandS5B
5 5
Onecaveatoftheselatterstudiesisthatthe4-HONH-biphenyl of the Supporting Information) when compared to the
used was not freshly prepared, and even in the presence of incubation controls (Figures 1A, 3A, and 5A), presumably
ascorbic acid, the disproportionation of 4-HONH-biphenyl because of nonenzymatic reduction of nitroso species. On the
(which also yields 4-ABP) was still significant under the contrary, P450 2S1 accelerated the process of disproportiona-
experimental conditions. tion (Figure 1C, and Figures S3C and S5C of the Supporting
Although most P450 reactions are oxidations,5 P450s can Information),andsomenonreductivepathwaysforthereaction
also catalyze a wide range of reductions,1 as summarized by of oxidized amines have been described previously.86
Wisloki et al.78 and more recently by Testa.79 Generally in the The nitroso derivatives of 5F 203 and 4-ABP were reduced
P450 catalytic cycle, the ferrous P450 species binds O tightly byNADPHwithNPRtoN-hydroxylamines(Figure7B)under
2
and oxidation occurs rapidly (after the addition of the second anaerobic conditions. In the presence of P450 2S1, as well as
electron).1However,intissueswhereonlyalowconcentration NPR, NO-5F 203 was reduced to 5F 203 (amine) almost
of O is present, e.g., in hypoxic cancer cells,80 reduction completely (Figure 3D). P450 2S1-catalyzed anaerobic
2
becomesmorefavorable.FerrousP450(E ∼−300mV)81,82 reduction of 4-HONH-biphenyl occurred but was not as
m,7
shouldbeareasonablygoodreducingagentifthesubstrateisin efficient as that with HONH-5F 203 (Figure 4D). A larger
an appropriately bound form and has the potential to accept amount of azoxybiphenyl (Figure 4D) compared to the
electrons. Alternatively, the binding of some substrates may incubation control (Figure 4A) is due to the fact that no
block(orpartiallyblock)theincorporationofO ,whichshould disproportionation can occur because no 4-HONH-biphenyl is
2
allowthereductiontooccurevenunderaerobicconditions.1It formed in the control reaction. Nitroso derivatives of aromatic
wasshownearlierthatAQ4N(adi-N-oxide)canbereducedby amines and HAAs can react with hemoglobin-β Cys-93 in the
both P450 2W1 and 2S1 under anaerobic conditions.51 bloodstream to form arylamine−hemoglobin sulfinamide
Subsequently, we found that 5F 203 can undergo P450 1A1- adducts in experimental animals.87,88 4-ABP has been reported
and 2W1-mediated bioactivation in the presence of O , and tohaveahighfractionofdose(>5%)boundtohemoglobinas
2
HONH-5F 203 (one of the reactive intermediates) can be sulfinamide adducts.89 Reduction of nitroso compounds may
reduced by P450 2S1 under anaerobic conditions and, to a decrease the level of formation of hemoglobin adducts. The
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nitrosoderivativeofIQhasbeenshowntobereducedbyNPR hydroxylamino-3-methylphenyl)-5-fluorobenzothiazole; IQ, 2-
to form the N-hydroxylamine and a trace amount of IQ.64 amino-3-methylimidazo[4,5-f]quinoline; LC−MS, liquid chro-
Similarly,thenitroderivativesofIQand2-NAarealsoreduced matography−mass spectrometry; mARC, mitochondrial ami-
byNPRtoN-hydroxylaminesasmajorproducts(Figure5A,C) doxime reducing component; MeIQx, 2-amino-3,8-
under anaerobic conditions. If P450 2S1 is also present, N- dimethylimidazo[4,5-f]quinoxaline; 2-NA, 2-naphthylamine;
hydroxylamines are reduced to yield IQ and 2-NA [amines NAT, N-acetyltransferase; NO, nitroso; NO-5F 203, 2-(3-
(Figure 5B,D)]. methyl-4-nitrosophenyl)-5-fluorobenzothiazole; NO , nitro;
2
In conclusion, our results indicate that human P450 2S1 is NPR, NADPH-P450 reductase; PhIP, 2-amino-1-methyl-6-
capable of catalyzing the reduction of N-hydroxylamine, phenylimidazo[4,5-b]pyridine; SULT, sulfotransferase.
■
nitroso, and nitro derivatives of at least several carcinogenic
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