Scavenging of Free-Radical Metabolites of Aniline Xenobiotics and Drugs by Amino Acid Derivatives

12 Pages·2013·1.73 MB·English

Article
pubs.acs.org/crt
Scavenging of Free-Radical Metabolites of Aniline Xenobiotics and
Drugs by Amino Acid Derivatives: Toxicological Implications of
Radical-Transfer Reactions
Karim Michail,*,†,‡ Argishti Baghdasarian,† Malyaj Narwaley,† Naif Aljuhani,†,§ and Arno G. Siraki†
†
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
‡
Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
§
Pharmacology and Toxicology Department, Faculty of Pharmacy, Taibah University, Madinah, Saudi Arabia
ABSTRACT: We investigated a novel scavenging mechanism of
arylamine free radicals by poly- and monoaminocarboxylates. Free
radicalsofarylaminexenobioticsanddrugsdidnotreactwithoxygen
in peroxidase-catalyzed reactions; however, they showed marked
oxygen uptake in the presence of an aminocarboxylate. These free-
radicalintermediateswereidentifiedusingthespintrap5,5-dimethyl-
1-pyrroline-N-oxide (DMPO) and electron paramagnetic resonance
(EPR) spectrometry. Diethylenetriaminepentaacetic acid (DTPA), a
polyaminocarboxylate, caused a concentration-dependent attenuation
of N-centered radicals produced by the peroxidative metabolism of
arylamines with the subsequent formation of secondary aliphatic
carbon-centered radicals stemming from the cosubstrate molecule.
Analogously, N,N-dimethylglycine (DMG) and N-methyliminodiace-
tate (MIDA), but not iminodiacetic acid (IDA), demonstrated a
similarscavengingeffectofarylamine-derivedfreeradicalsinahorseradishperoxidase/H O system.Usinghumanpromyelocytic
2 2
leukemia (HL-60) cell lysate as a model of human neutrophils, DTPA, MIDA, and DMG readily reduced anilinium cation
radicals derived from the arylamines and gave rise to the corresponding carbon radicals. The rate of peroxidase-triggered
polymerization of aniline was studied as a measure of nitrogen-radical scavenging. Although, IDA had no effect on the rate of
aniline polymerization, this was almost nullified in the presence of DTPA and MIDA at half of the molar concentration of the
anilinesubstrate,whereasa20molarexcessofDMPOcausedonlyapartialinhibition.Furthermore,theyieldofformaldehyde,a
specific reaction endproduct of the oxidation of aminocarboxylates by aniline free-radical metabolites, was quantitatively
determined. Azobenzene, a specific reaction product of peroxidase-catalyzed free-radical dimerization of aniline, was fully
abrogated inthepresenceofDTPA,asconfirmedbyGC/MS.Underaerobicconditions,aradical-transfer reactionisproposed
between aminocarboxylates and arylamine free radicals via the carboxylic group-linked tertiary nitrogen of the deprotonated
aminoacidderivatives.Thesefindingsmayhavesignificantimplicationsforthebiologicalfateofarylaminexenobioticanddrug
free-radical metabolites.
■
INTRODUCTION carboxylic acid analogues of those compounds (polyamino-
carboxylates and their derivatives) with the peroxidase-
Arylamine compounds are represented by important classes of
mediated free-radical metabolism of aromatic amines.
xenobiotics, which include drugs, carcinogens, and environ-
Aminopolycarboxylic acid metal-chelating agents (e.g., ethyl-
mental pollutants. The peroxidase-driven metabolism of
enediaminetetraacetic acid, EDTA, Figure 1) are amino acid-
arylamine xenobiotics, such as aniline (ANL, Figure 1), and
like organic compounds that have important industrial,
ANL-based drugs, such as aminoglutethimide (AG, Figure 1),
agrochemical, and biomedical applications. EDTA was first
has been studied by us and others and was shown to produce
synthesized in the early 1930s.4 An extended EDTA molecule,
nitrogen-centeredanilinium(orcation)radicalmetabolitesthat
spontaneously split into secondary phenyl radicals.1,2 Recent diethylenetriaminepentaacetic acid (DTPA, Figure 1), is
data from our laboratory demonstrated that, in biochemical another well-known metal chelator of widespread use, which
peroxidase-catalyzedsystemsusingplantorhumanperoxidases forms stable complex ions with transition-metal cations and
(i.e., horseradish peroxidase (HRP) or myeloperoxidase, alters the oxidation−reduction properties of transition heavy-
respectively), biological and synthetic polyamines synergisti- metal ions. From another standpoint, because other metal
callyenhancedthegenerationofN-centeredradicalmetabolites
derivedfromaromaticaminedrugsorxenobiotics.3Inthesame Received: July4, 2013
vein, we have investigated the redox interaction of the Published: November 5, 2013
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Figure1.Molecularstructuresofxenobiotics,drugs,standards,polyaminocarboxylates,dicarboxylicacids,aminoacids,aminoacidderivatives,and
monoaminocaboxylates involved in this study.
chelators such as deferoxamine possess antioxidant capacity, it wereoxidizedbyfree-radicalmetabolitesofaromaticamines.In
is conceivable that DTPA could also have some degree of addition,aminocarboxylicacidderivativesthathadasignificant
antioxidant activity. For instance, it has been proved that propensity to form secondary, reducing carbon-centered
aminocarboxylic acid ligands (e.g., EDTA) as well as their radicals exhibited different biochemical properties than the
phosphonicacidanalogues(e.g.,N,N,N′,N′-ethylenediaminete- precursorANLfree-radicalmetabolites.InadditiontoEPRspin
trakis-(methylenephosphonic)acid)possess,besidestheirwell- trapping,wealsoperformedoxygenuptake,colorimetricassays,
documentedmetal-sequesteringactivity,potentradical-scaveng- kinetic spectrophotometric measurements, and gas chromatog-
ingproperties, as evidencedby their diffusion-limited reactions raphy−mass spectrometry (GC/MS) to investigate the
with hydroxyl radical (•OH) at steady-state radical concen- mechanism of the interaction between polyaminocarboxylates
trations.5Cabelli-Bielskiandothershaveextensivelystudiedthe and N-centered free-radical metabolites of ANL substrates
rate constants for the reactions of DTPA with hydroxyl (k = 2 generated by horseradish peroxidase or human promyelocytic
×109M−1s−1)andcarbonateradicals(k=1.7×107M−1s−1); ■leukemia cell lysate and hydrogen peroxide.
these are catalogued at the National Institute of Standards and
Technology (NIST).6,7 In addition, DTPA and similar MATERIALS AND METHODS
chelators have been reported to react with 2,2′-azinobis(3- 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) from Cedarlane Labora-
ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, tories(manufacturedbyDojindoMolecularTechnologies,Inc.,Japan)
peroxynitrite,andhypochlorite.8Thus,itislikelythatreactions was stored under an argon atmosphere at −80 °C and was used
without further purification. AG was purchased from Toronto
between other biologically important reactive radicals and
Research Chemicals (Toronto, ON). Sodium phosphate buffer (0.1
polyaminocarboxylates are also feasible, where the latter would
M, pH 7.4) (PB) was prepared and used after metal chelation by
function as electron donors. DTPA is used at varying incubating overnight at 4 °C with Chelex-100 resin (Bio-Rad
concentrationsinbiochemicalsystems,butinEPRexperiments Laboratories, Mississauga, ON). ANL and its derivatives (>99.5%),
it is typically used at a concentration range between 0.1 and 1 viz.4-chloroaniline(4-CA,Figure1)andp-toluidine(TOL,Figure1),
mM.9Sincethelate70sandearly80s,ithasbeendemonstrated TraceSELECT hydrogen peroxide (HO) 30% v/v (Fluka, Buchs,
2 2
that the use of DTPA in EPR studies minimizes radical Switzerland), analytical grade DTPA (Fluka, Buchs, Switzerland),
formation that is ascribed to trace-metal-catalyzed oxidation molecular biology tested EDTA, glycine (99%) (Figure 1), DMG
reactions.10 (Figure 1) (≥99%),L-asparticacid (98%)(Figure 1),L-glutamicacid
(99.5%)(Figure1),glutaricacid(GA,Figure1)(99%),IDA(Figure
To the best of our knowledge, aminocarboxylates have not
1) (98%), E-azobenzene (AZO, Figure 1) (99%), horseradish
beforehand been investigated for their scavenging potential of peroxidase (HRP) type VI (activity: 250−330 units/mg), protease
free-radical metabolites of ANL compounds in peroxidase
inhibitorcocktail,andchromotropicaciddisodiumsaltdihydrate(CA)
systems. We employed EPR spin-trapping experiments to (≥99%) were purchased from Sigma-Aldrich (Oakville, ON). Gibco
characterize a number of aminocarboxylic acid derivatives that RPMI1640mediumwithL-glutamine,antibiotic−antimycoticmixture
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(10000units/mLofpenicillin,10000μg/mLofstreptomycin,and25 NanoDrop 2000c software for recording and data acquisition. All
μg/mL of amphotericin B), and fetal bovine serum were obtained measurements were made at least in triplicate.
from Invitrogen-Life Technologies. MIDA (Figure 1) (99%; Alfa ColorimetricAssay.Biochemicalreactionmixtures(finalvolumes
Aesar) was purchased from VWR. All other reagents and materials 200μL)containing5mMANLorAGinthepresenceandabsenceof
were of commercially available grades. Stock solutions of DTPA (50 equimolarconcentrationofDTPA,MIDA,orDMGwerepreparedas
mM)werepreparedbydissolvingappropriatequantitiesoftheacidin described for the EPR study experiments using 5 μM HRP and 500
0.5 M NaOH to yield a solution with a final pH of ∼6.5. HO was μMHO.Reactionswereincubatedfor10minatrt,andtheyieldof
assayedusingitsextinctioncoefficient(43.6M−1cm−1)at2420n2m.11 formal2deh2ydeineachreactionwasdeterminedusingCA.Briefly,100
Allother reagents used were of the best available commercial grade. μLofCAwasaddedto50μLofthereactionmixturethatwasheated
Cell Culture. HL-60 human promyelocytic leukemia cells were at70°Cfor20min.Thesolutionwasallowedtocoolatrtfor5min,
obtainedfromATCC(catalogno.CCL-240,Manassas,VA)andwere 350μLofwaterwasadded,anda450μLaliquotofthepurple-colored
cultured in RPMI 1640 medium supplemented with 10% heat- solution was measured at 580 nm using a Thermo Scientific
inactivated fetal bovine serum and 1% Gibco antibiotic−antimycotic NanoDrop 2000c dual-mode UV−vis spectrophotometer.
mixture.Cellsweremaintainedinamoistatmospherewith5%CO at All measurements were made in triplicate, and quantitative
2
37°Candwereallowedtogrowthroughsevenpassagesbeforelysis. determinations of the yield of CHOin each reaction were based on
Cells(7×106cells/mL)wereharvestedbyspinningat120gfor5min, a standard curve (r2 = 0.99) 2constructed using five different
and the cell pellet was washed by two volumes of cold phosphate- concentrations of the analyte in the range of 0−80μM.
buffered saline and lysed using cold RIPA buffer (1% sodium Gas Chromatography/Mass Spectrometric Analysis. Bio-
deoxycholate(w/v),1%TritonX-100(v/v),and0.1%SDS(w/v)in chemical reaction mixtures containing ANL in the presence and
PBS) containing 0.01% (v/v) protease inhibitor cocktail, which was absence of DTPA were prepared as described for the EPR study
addedimmediatelybeforeuse.Aftersonicationfor30sat50%pulse experiments, and reaction products were isolated by liquid−liquid
and centrifugation (15000g) at 4 °C for 15 min, the cleared extractionbyshakingwith500μLofhexanefor5min.Liquid−liquid
supernatant was isolated for usein EPR experiments. extracts together with a standard solution of AZO at 50 μM final
Oxygen-Consumption Analysis. Oxygen consumption was concentration were sampled and run in an Agilent 5975C Series GC
recorded using an YSI 5300 biological oxygen monitor (Yellow coupled with a single quadrupole MS detector in EI mode using a
SpringsInc.,YellowSprings,OH)withaClarkoxygenelectrode.The programmable ion source temperature (50−290 °C/5 min at 10 °C
data was recorded using a DATAQ interface (Akron, OH), which increments). Full-scan MS data was acquired using ChemStation
allowedforaWindows-basedPCtomonitortheoxygenconsumption E.01.00MSDsoftwareandsearchedagainsttheNIST08MSspectral
in each sample. Rates of oxygen consumption from at least three library. The yield of AZO from each reaction was computed by
independentexperimentswerecalculatedafterthedatawasimported comparisontothestandardsolution.Allmeasurementsweremadeat
intoMicrosoft Excel. least in duplicate.
■
All reactions were carried out in PB (0.1 M, pH 7.4) at room
temperature.Theycontained5mMaromaticamineand1mMHO
andwereinitiatedby1μMHRPintheabsenceorpresenceof1m2M2 RESULTS
DTPA. Oximetric Detection of DTPA Co-oxidation by ANL-
Electron Paramagnetic Resonance (EPR) Study. Reactions Derived Free Radicals. As is apparent from Figure 2,
(200μLfinalvolumes)werecarriedoutinChelex-100-treatedPB(0.1
arylamine xenobiotics and drugs apart from TOL (Figure 2A)
M,pH7.4)atvariableconcentrationsofDTPArangingfrom0.05to6
mMcontaining1mMANLorAG,100mMDMPO,20μMHRP,and didnotinducedetectableoxygenconsumptioninthepresence
400 μM HO added last to trigger the reaction and were run in an of HRP/H2O2 (Figure 2B−D). Control reactions containing
2 2
Elexsys E500 EPR spectrometer using the following instrumental DTPA, a representative example of the studied amino acid
settings: microwave frequency, 9.8 GHz; incident microwave power,
20.0mW;centerfield,3485G;scanrange,100.0G;fieldmodulation,
1.0G;receivergain,60.0dB;andtimeconstant,167.8ms.Individual
reactions were repeated at least induplicate on nonconsecutive days.
DTPA, EDTA, and substructural congeners, such as amino acids,
substitutedaminoacids,dicarboxylicacids,andamines(GA,Gly,Asp,
IDA,MIDA,andDMG)wereinvestigatedbyreplacingDTPAinthe
aforementioned reactions. Controls not containing the investigated
aminocarboxylatesorthestudiedsubstrates(ANLorAG)wererunin
parallel. After a brief vortex-mixing, the reaction mixture was
transferred to aflat cellfor data acquisition.
Similarlypreparedreactionswerestudiedusing10μLofHL-60cell
lysatecorrespondingto7×106cells/mLandcontained5mMANLor
AGinthepresenceorabsenceofDTPAorMIDAathalfofthemolar
concentration of the substrates. Reactions were triggered by 50 μM
HO.
2 2
TheobtainedEPRspectraweresimulatedanddeconvolutedusing
WinSim version 0.98 obtained from the Public EPR Software Tools
(National Institute of Environmental Health Sciences, NIH). Figure 2. Oxygen consumption catalyzed by oxidation of DTPA by
Kinetic Spectrophotometry. Biochemical reactions containing different arylamine xenobiotics and drugs in the presence of HRP/
ANL (2 mM), HRP (0.1 μM), and HO (200 μM) in the presence HO. Reactions that contained 5 mM aromatic amine, 1 μM HRP,
2 2 2 2
and absence of different aminocarboxylates at 1 mM, namely, IDA, and1mMHO without(dashedlines,A−E)orwith(solidlines,F−
2 2
MIDA,andDTPA,werepreparedasdescribedintheprevioussection I)1mMDTPAwererunasdescribedinthetextandweretriggered
(EPRstudy).Resultswerecomparedtoacontrolreactioncontaining by the peroxidase (arrow). In the absence of DTPA, only TOL (A)
100 mM of the spin trap, DMPO. As soon as the reaction was showedoxygenuptake,butotherarylamines,namely,4-CA(B),ANL
triggered using HO, it was followed spectrophotmetrically in the (C), AG (D), and vehicle (E), were not able to catalyze oxygen
2 2
kineticmodeat450nmfor15minat1minintervalsusingaThermo consumption.InthepresenceofDTPA,significantoxygenuptakewas
Scientific NanoDrop 2000c dual-mode UV−vis spectrophotometer observedinreactionscontaining4-CA(F),ANL(G),TOL(H),and
(Wilmington, DE) connected to a desktop PC supplied with AG (I).
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Figure 3. Concentration-dependent modifications of the EPR spectrum of ANL-derived spin adduct (ANL/DMPO•) by DTPA. Reactions were
carriedoutinChelex-100−treatedphosphatebuffer(0.1M,pH7.4).TheEPRspectraresultingfromreactionscontaining1mMANL,100mM
DMPO,400μMHO,and20μMHRPwithoutDTPA(A),with0.05mMDTPA(B),with0.1mMDTPA(C),with0.3mMDTPA(D),with0.6
2 2
mMDTPA(E),andwith0.9mMDTPA(F)areshown.TheomissionofANLfromreactionFshowednosignificantspectrum(G).Thespectrum
ofreactionAwassimulated(H,r2=0.88),whichrevealedthehyperfinesplittingconstantsofatrappedANLradical:aN(nitroxide)=15.5G,aH=
18.2G,andaN=2.2G.ThespectruminFwassimulated(I,r2=0.98),whichrevealedacompositespectrumcontainingtheANL-derivedradical
species fromreaction A and aDTPA-derived species (aN= 15.8 G andaH= 21.8 G).
derivatives, HRP, and H O in the absence of an arylamine peroxidation of 4-CA (Figure 2F) resulted in the highest
2 2
substratealsodidnotshowoxygenuptake(Figure2E).Infact, oxygen consumption followedbyANL(Figure2G)thenTOL
the presence of DTPA was a prerequisite, which catalyzed (Figure 2H) and finally AG (Figure 2I), apparently because of
oxygen uptake subsequent to the peroxidation of the studied the generation of radical metabolites from these ANL
ANL-based compounds and the production of ANL-derived derivatives, which presumably oxidized the aminocarboxylate
precursor free-radical metabolites except for TOL; the latter moleculeandresultedintheproduction ofnoticeablydifferent
showed subtle oxygen uptake in the absence of DTPA after daughter radical species that were able to reduce (scavenge)
catalyzingthereactionwithHRP/H O .However,theamount molecular dioxygen in aqueous solution at neutral pH (Figure
2 2
of consumed oxygen was considerably less than when both 2F−I).
DTPAandthesubstratewereconcomitantlypresent(Figure2, TocorrelatetheextentofDTPA-enhancedoxygenuptakeof
panel A vs H). In the presence of DTPA, the enzymatic free-radical metabolites of ANL-based compounds with the
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nature,yield,andratioofthosereactive species,wecarriedout
EPR experiments to characterize primary and secondary
radicals involved in the free-radical interaction.
Identification of Arylamine Free-Radical Metabolites
and Reactive Intermediates of Amino Acid Derivatives
by EPR. We first performed EPR experiments using different
concentrations of DTPA to estimate the scavenging effect by
the chelator molecule on ANL-derived free-radical metabolites
that were trapped by the cyclic nitrone-type spin trap, DMPO.
In the absence of DTPA, the enzymatic peroxidation of ANL
yielded a typical EPR spectrum consisting of a doublet of
triplets that was further split into triplets that could be
attributed to the sum of the interactions of the unpaired
electronwiththethreeprotonsofnuclearspin1/2(3×Hβ)in
DMPO and with the 14N nucleus of nuclear spin 1, giving rise
to a total of 18 lines. The EPR spectrum was confirmed by
simulation that connotes an anilinium-type radical species that
isinequilibriumwiththeunchargedanilinoradicals(Ar-NH +• Figure 4. DTPA scavenging of the parent N-centered cation radical
2 metabolites of AG catalyzed by HRP/HO. Reactions were carried
⇌ Ar-NH•) (Figure 3A,H). The weak signals obtained outinChelex-100-treatedphosphatebuffe2r(20.1M,pH7.4).Reactions
implicitly suggest that the flux of anilinium radicals is quite containing 1 mM AG, 100 mM DMPO, 400 μM HO, and 20 μM
2 2
low. However, DTPA caused concentration-dependent mod- HRPwithoutDTPA(A),with0.9mMDTPA(B),with6mMDTPA
ifications to the spectra of parent ANL-derived free-radical (C),andwithnoAGand6mMDTPA(D)areshown.Withspectrum
metabolites, resulting in a composite EPR spectrum consisting Cbeingthemostintense,itwasscaledby0.25,andwhensimulated,it
oftwodistinctparamagneticspecies,viz.nitrogen-andcarbon- showedthefollowinghyperfinesplittingconstants:aN=15.8GandaH
= 22.1 G (E,r2= 0.99).
centered radicals originating fromthe ANL substrate andfrom
the aminocarboxylate, respectively (Figure 3B−E). At the
highestconcentrationofDTPA(0.9mM),aprominentsix-line carboxylic acids, such as AA, annihilated the paramagnetic
EPR spectrum could be detected, which was almost entirely spectrum of ANL’s N-centered radicals (Figure 5C). Analo-
due to DTPA-derived free radicals (Figure 3F). Importantly, gously, GA, a dicarboxylic acid, exerted the same effect (data
DTPA was unable to generate any free radicals in the absence not shown). However, dicarboxylic amino acids, such as Asp,
ofaconcomitantaromaticaminesubstrateatanyofthestudied didnotaffectthenativenitrogenspectrumoftheanilinoradical
concentrations (Figure 3G). Simulation of spectrum F (Figure cations (Figure 5D). Likewise, Glu, which has one more
3) involving the most significant contribution from DTPA methylene group than Asp, showed a comparable behavior
revealedacompositespectrumthat,asidefromtheN-centered (data not shown). Furthermore, monocarboxylic amino acids,
cation radical metabolites, revealed the formation of a new such as Gly, caused only a subtle attenuation of ANL-derived
speciesthatwascharacterizedasaC-centeredradicalidentified primary free radicals (Figure 5E). However, DMG, an N-
by hyperfine splitting constants (hfsc) aN = 15.8 G and aH = methylated glycine having a tertiary amino function, promoted
21.8GandcanbeattributedtoDMPO/DTPAradical(Figure the conversion of precursor ANL-derived N-centered radical
3I). Similarly, in the absence of DTPA, the ANL-based drug metabolites into daughter C-centered radicals (Figure 5F).
(AG) under the given experimental conditions gave rise to an Furthermore, MIDA, but not IDA (Figure 5, panels G and H,
asymmetric spectrum showing multiple free-radical species respectively) demonstrated the same behavior as DTPA,
(Figure4A).Nonetheless,whenDTPAwasadded(0.9mM),it EDTA, and DMG and was able to scavenge anilinium cation
altered the original EPR spectrum of the drug substantially radicalmetabolitesofANL,producingcarbonradicals.Control
(Figure 4B). Furthermore, at higher levels (6 mM), a reactions lacking an arylamine substrate but containing the
completelynewandintensespectrumappearedthatwassolely aforementioned molecules that were readily oxidized by ANL-
due to secondary carbon-centered free radicals derived from derived free radicals did not produce a detectable EPR
DTPA (Figure 4C). Importantly, oxidation of DTPA at this spectrum with HRP/H O (data not shown).
2 2
concentration by HRP/H O showed a minimal paramagnetic Finally, we aimed at testing whether the aforementioned
2 2
spectrum (Figure 4D). A simulated spectrum of the findings regarding the described radical-transfer reactions
experimental spectrum in Figure 3C using the hfsc of aN between ANL free-radical metabolites and the studied amino
(nitroxide) = 15.8 G and aH = 21.8 (r = 0.99) is shown in acid derivatives could be of biological significance. For that
Figure 4E. purpose,weinvestigatedthepossibilityofthatinteractionusing
Next,weusedEDTA,ametalchelatorstructurallyrelatedto HL-60 cell lysate. Indeed, similar results were obtained either
DTPA, and several substructural congeners of DTPA, namely, using ANL or AG as substrates, where DTPA (Figure 6B,F)
acetic and glutaric acids as organic mono- and dicarboxylic and MIDA (Figure 6C,G) but not IDA (Figure 6D) were
acids,respectively,glycineandglutamic/asparticacidsasmono- capable of reducing anilinium free-radical metabolites and
and dicarboxylic amino acids, respectively, and DMG as a generating carbon-centered radicals compared to control
methylated amino acid. In addition, we also tested MIDA and reactions not containing the amino acid derivative (Figure
IDA. In peroxidase-initiated biochemical reactions, EDTA 6A,E).
behaved similarly to DTPA and altered the 18-line EPR Quantification of the Reaction End Product of the
spectrum of ANL-derived radical metabolites (Figure 5A) to a Oxidation of Amino Acid Derivatives by ANL Free-
6-line spectrum because of the formation of carbon-centered RadicalMetabolites.Theamountofformaldehydeformedin
daughter radicals (Figure 5B). In contrast, organic mono- each reaction was quantitatively determined with no prior
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Figure 6. Metabolic bioactivation of ANL and AG into reactive
intermediatesusingHL-60celllysateandelectron-transferreactionsto
Figure 5. Effect of DTPA-related polyaminocarboxylic acid metal
theaminoacidderivativesDTPAandMIDA..Reactionscontaining5
chelators,mono-anddicarboxylicacids,mono-anddicarboxylicamino mM ANL (A−D) or 5 mM AG (E−G) in the absence (A and E,
acids, or amino acid derivatives on line shapes, magnitudes, and respectively)orpresenceofDTPA(BandF,respectively),MIDA(C
hyperfine splitting patterns of the EPR spectra of DMPO-trapped and G, respectively), or IDA (D) were carried out in Chelex-100-
ANL-derived N-centered radicals (DMPO•/NH-Ar) catalyzed by treatedphosphatebuffer(0.1M,pH7.4)andwerecatalyzedbyHL-60
HseRttiPn/gHs2Owe2.reBiothcohseemidcaelscrreibacetdionincotnhdeititoenxts.a(nAd−EHP)RNspaetcivtreosAcoNpLy cell lysate corresponding to 325000 cells/mL and50 μM H2O2.
spectrum representing basal level DMPO-trapped nitrogen radicals
addition reactions of monomeric ANL molecules was followed
before (A) and after addition of 5 mM of EDTA (B), AA (C), Asp
(D), Gly (E), DMG (F), MIDA (G), or IDA (H). The hyperfine kinetically by spectrophotometric measurement of the reaction
splittingconstants(hfsc)inaqueousphosphatebuffer,pH7.4,atroom products at λmax450nmasafunctionoftime.The free-radical
temperaturewereaN(nitroxide)=15.6G,aH=18.0,andaN(ANL)= oligo-/polymerizationofANL,inthepresenceandtheabsence
2.14Gforthenitrogen-radicalspeciesinpanelsA,D,E,andH,andaN of DTPA, MIDA, IDA, or DMPO, was initiated by catalytic
(nitroxide) = 15.8 G and aH = 21.8 for the carbon-radical species in concentrationsofHRP/H O .Thevisibleabsorbanceofdimers
2 2
panels B, F, and G. Note that panels B, F, and G, showing the and/or oligomers/polymers of ANL monitored at the selected
formation of C-centered free radicals, were scaled to eachother. wavelength were almost fully attenuated in the presence of
DTPA compared to control reactions containing no amino-
sample preparation steps using chromotropic acid that carboxylates (Figure 8, panel A vs E, respectively). Similarly,
selectively reacted with formaldehyde, giving rise to a colored MIDA considerably inhibited the formation of ANL-derived
adduct that could be traced colorimetrically (Figure 7A−D). azo-polymers(p=4.3×10−8)asdemonstratedbyasignificant
Using either ANL or AG as a peroxidase donor substrate, decrease in the extent of light absorption at 450 nm over time
formaldehyde was a major end product of the reaction in the compared to the control reaction (Figure 8, panel A vs D).
cases of MIDA and DMG, whereas its yield was comparatively However, IDA did not affect the rate of ANL polymerization
lowerinthecaseofDTPA.Forexample,inthecaseofAG,the (Figure 8B), whereas DMPO at a concentration of 100 mM
yieldofformaldehydeexpressedinmicromolespermilligramof had a moderate inhibitory effect on ANL polymerization. In
HRPproteinwasinthefollowingorder:MIDA(115.9±0.03) addition, the suppression of the rate of generation of the
> DMG (89.4 ± 0.02) ≫ DTPA (7.8 ± 0.01). colored products monitored at 450 nm was significantly
Kinetic Spectrophotometric Monitoring of the Effect different statistically when comparing MIDA at one-half of
of Aminocarboxylates on the Polymerization of ANL. the molar concentration of the ANL substrate to a 50 molar
ThereactionprogressofANLoligo-/polymerizationbyvarious excess of DMPO (p = 5.1 × 10−6). Furthermore, although 10
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Determination of the Extent of Inhibition of Free-
Radical ANL Polymerization by Polyaminocarboxylates
using GC/MS. The dimer of ANL, azobenzene (AZO), was
selectivelyextractedinhexaneandchromatographedusingGC
inconjunctionwithEI-MSdetection.Theextractionprocedure
wasverifiedtobefreefrominterferenceduetothepresenceof
DTPAbycomparingtheUVandvisibleabsorbancereadingsat
two different wavelengths (320 and440 nm)ofstandard AZO
(0.1 mM) extracted in hexane from aqueous mixtures in the
absence or presence of a 50 molar excess of DTPA (5 mM).
DTPA did not show any significant effects on the extraction
efficiencyofAZO(datanotshown).Thepeakat14.9minwas
identified as AZO by R (chromatographic retention data)
t
matchingwithastandardandcomparisonwithareferenceMS
spectral database (Figure 9, panels C and A, respectively).
Figure 7. Colorimetric quantification of formaldehyde, a specific
reaction end product of the oxidation of amino acid derivatives by
ANL free-radical metabolites. Biochemical reactions (200 μL) were
carried out in Chelex-100-treated phosphate buffer (0.1 M, pH 7.4)
andcontained5mMAG(AandC)orANL(B),5μMHRP,and500
μMHO inthepresenceof5mMDTPA(A)or5mMMIDA(Band
2 2
C) after reacting an aliquot of the reaction mixture (50 μL) with
chromotropic acid/sulfuric acid as indicated in the text. The yield of
formaldehyde in each reaction was compared to standard calibration
reactions containingdifferent concentrations of formaldehyde instead
of the arylamine substrate, of which the 80 μM calibration level is
displayed in spectrumD.
Figure9.DTPAabrogatestheformationofthemetabolicendproduct
of the one-electron oxidation of ANL by HRP/HO. GC/MS
2 2
chromatograms of azobenzene standard (panel A) vs extract of
peroxidase-catalyzed oxidation reactions of ANL with (panel B) and
without(panelC)DTPAinpresenceofHO accordingtothestated
2 2
conditionsinthetext.Asshown,thepresenceofDTPAinhibitedthe
formation of azobenzene.
When DTPA, a model compound for the studied amino acid
derivatives, was present in the biochemical reaction mixture at
an equimolar concentration as that of the ANL substrate, the
Figure8.Effectofmono-andpolyaminocarboxylicacidsontherateof AZO peak totally disappeared from the gas chromatogram
oligo-/polymerization of ANL catalyzed by HRP/HO. Biochemical (Figure 9B).
2 2 ■
reactions containing ANL (2 mM), HRP (0.1 μM), and HO (200
2 2
μM) in the absence (A) or presence of 1 mM IDA (B), 100 mM DISCUSSION
DMPO(C),1mMMIDA(D),or1mMDTPA(E)werekinetically
followed for 15 min by spectrophotometric measurements at λ 450 ANLanditsderivatives,whichshareanarylaminesubstructure,
nm. The rate of formation and yields of the colored oligomers or are organic compounds of widespread use in the industry and
polymersweresignificantlysuppressedinthepresenceofeitherMIDA manufacturing of several agrochemicals and pharmaceuticals.
orDTPAatone-halfofthemolarconcentrationoftheANLsubstrate However, they are also considered universal pollutants as
(D and E, respectively) but only moderately suppressed in the
globally occurring xenobiotics andindustrial waste, which pose
presenceof a50 molarexcess of DMPO(C)when comparedto the a considerable environmental and occupational health risk.12,13
control reaction (A). IDA had no effect on the rate of ANL oligo-/
ANL-based drugs are not devoid of life-threatening adverse
polymerization (B).
effects, as they are strongly implicated with blood dyscrasias
that are likely to predispose an individual to a compromised
mM DMPO (i.e., a 5 molar excess compared to the ANL immune status, making the body incapable of combating
substrate) resulted in some inhibition of ANL polymerization, microbial infections and hence more susceptible to fatal
this was not significantly different from the rate of peroxidase- septicemia even by endogenous microbial flora.14 Neutrophils
catalyzed ANL polymerization in the absence of DMPO (p = (polymorphonuclear leucocytes), which predominate the
0.36; data not shown). leucocyte pool, are abundant in myeloperoxidase, an immune
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Chemical Research in Toxicology Article
specificperoxidasethatproducesaninsitupotentbactericidal/ schematically represented by Armstrong et al. and Surdhar et
fungicidal agent, hypochlorous acid (HOCl). However, the al.forthestructurallyrelatedcompoundEDTA.23,24Finally,an
other side of the coin is that this very same heme protein is a expected intramolecular rearrangement is supposed to occur
potent generator of free-radical metabolites from ANL-based through migration of a hydrogen atom from the α-carbon to
drugs, a mechanism that has been postulated to induce the decarboxylated primary carbon harboring the free electron
•
idiosyncratic drug reactions, such as agranulocytosis (i.e., ( CH ), culminating in the formation of the more thermody-
2
depletion of blood neutrophil count).15 For example, AG, a namicallystablesecondarycarbonradical(•CH),whichreacted
first-generation aromatase inhibitor, was shown to repeatedly rapidly with oxygen in the described experimental setting
induce idiosyncratic neutropenia in vivo.16 (Figure 2G,I). Interestingly, the latter has been demonstrated
Oxygen analysisdemonstrated thatthepresenceofelectron- to be a major long-lived radical under anaerobic con-
withdrawing groups on the ANL ring, such as a chloro (−Cl) ditions.6,19,23,24
substituent, enhances the oxidation of the aminocarboxylate Most importantly, DTPA proved to be a weak peroxidase
moleculeprimarilybecauseofahighoxidationpotential(E = substrate (on the basis of EPR spin trapping), as did other
ox
0.625V)thatisgovernedbythedegreeofacidityoftheaniline related molecules and substructural congeners because they
radicalcation.Thecationradicalof2-CA(pKAr−NH2+•=3.9)is werenotmetabolizedbyHRP(Figures3Gand4D);however,
more acidic than one derived from TOL (pKAr−NH2+• = 8.5). they indeed underwent a rapid reaction as excellent electron
(Figure2F).17Infact,theoxidationpotentialisonlyminimally donors with a concomitant aromatic amine substrate by
affected by the radical-stabilizing/destabilizing effect (electron- scavenging ANL one-electron oxidation product(s) (Figures
withdrawing substituents stabilize the parent aniline but 3F, 4C, and 5B,F,G).
destabilize the ensuing radical cation, such as the 2-Cl group We aimed at deciphering the structural requirements for an
in 2-CA, whereas the opposite is true for electron-donating aminocarboxylate molecule that would enable it to donate
substituents, such as the methyl group in TOL). However, readily an electron to free-radical metabolites stemming from
electron-donating substituents, such as a methyl (−CH ) ANLxenobioticsanddrugsandultimatelytogenerateauxiliary
3
substituent, that possess a hyperconjugative effect appear to free-radicalspeciesderivedfromtheaminocarboxylate.Initially,
contribute less efficiently to the electrophilic attack of ANL monovalent anddivalent organic acids werestudied to testthe
radicalsonDTPA,atleastinpart,becauseofaloweroxidation redoxreactivityofthe−COOHfunctionalgroupinDTPAand
potentialdrivenbyahigherbasicitycomparedto2-CA(Figure similar scavengers toward anilinium radicals; however, the
2H).17 In summary, the rate of oxygen uptake correlated with metabolism of anilines was absolutely prohibited in the
the extent of the acidity of the radical cation and the presence of weak carboxylic acids at the levels used in this
corresponding redox potential rather than the stabilizing/ study.Ionizableprotons,suchasthosecontainedinmono-and
destabilizing effect of the substituent through either spin diprotic organic acids including acetic and glutaric acids,
delocalization or polar effects.18 These results are also in good enhance,supposedly,ion-pairformationwithANL,aweakbase
agreement with previous reports showing that under aerobic that has a pK value of 4.6, suppressing the generation of free-
a
conditions EDTA-derived carbon-centered radicals undergo a radical metabolites. Aside from being of a less complex
fast reaction with oxygen.19 structure, Gly, the simplest α-amino acid, differs from EDTA
EPRspectroscopy isthe primary andmostdirect method to in having a protonated nitrogen at pH 7. Given that Gly has
detect and to characterize free radicals initially. Typically, it is been shown to undergo facile decarboxylation by hydroxyl
coupled to a spin-trapping step that precedes the actual radicals in aqueous solution, it is plausible that some of the
spectroscopic measurement. Spin trapping is defined as the produced anilinium radicals that have a relatively high redox
process of scavenging a short-lived free-radical intermediate potential (∼1 V for Ar-NH +•/Ar-NH ) interacted to some
2 2
using a diamagnetic agent and turning it into a relatively long- extent with Gly through hydrogen abstraction from the amino
lived paramagnetic product or spin adduct that can yield acid, which is characterized by an exclusively labile C−H
generic information about the identity and nature of the initial bond.24−27Thesubtlereduction inthe intensityofperoxidase-
radicaltrappedbydictatingaspecifichyperfinesplittingpattern initiated ANL radicals in the presence of glycine might be
and magnitude.20,21 The spin trap DMPO is exquisitely suited attributable to the aforementioned factors (Figure 5E).
to trap free radicals in a biological milieu or in an in vitro Nevertheless,otherpossibleexplanations,suchasaninteraction
cellular system because of its low toxicity and versatility. with the peroxidase enzyme or the ionization of ANL, cannot
Utilizing EPR spin trapping, we pursued the elucidation of the be excluded. The fact that onlyDMG and not Gly was able to
reaction mechanism by which the studied amino acid scavenge anilinium cation radicals efficiently led us to
derivatives scavenged anilinium cation radicals (Figures 3 and hypothesize that an essential structural prerequisite for an
4). With polyaminocarboxylates (e.g., DTPA), this could be auxiliarymoleculetointeractwithANLfree-radicalmetabolites
rationalizedbyconsideringanelectrontransferfromthereadily istheavailabilityofatleastonetertiarynitrogeninthechemical
available lone pair of electrons of one of the two equivalent radical-trap molecule. To confirm this notion, we tested other
“peripheral” tertiary amine nitrogens in the triamino acid compounds typifying a simplified structure of DTPA, such as
DTPA to the electrophilic anilinium radical, yielding a DTPA MIDAandIDAwhichpossesstertiaryandsecondarynitrogens,
radicalintermediatethatisstabilizedbyelectrondelocalization, respectively;asanticipated,onlyMIDAdemonstratedthesame
presumably over the nitrogen centers. Given that DTPA has behavior as DTPA, EDTA, and DMG, whereas IDA did not
five discernible pK’s, this intermediate will be present in significantly interact with free-radical metabolites of the ANL
a
solution in one or more of its corresponding prototropic molecule but only slightly mitigated those radical metabolites,
conjugates depending on the pH.22 Next, an efficient probably by affecting the deprotonation of such a weakly basic
decarboxylation step favored by the formation of a six- substrate.
membered-ring intermediate takes place, giving rise to a A previously published work by our group showed that
decarboxylated DTPA radical, a pathway that has been identical biochemical systems containing ethylenediamine
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Chemical Research in Toxicology Article
Scheme 1. Proposed Mechanistic Pathway for the Interaction between Aminocarboxylates and Arylamine Free-Radical
Metabolites
(EDA),aprimaryaminethatstructurallyconstitutesthecentral (Figure 7). Accordingly, di- or polyaminocarboxylates (viz.
part in DTPA molecule, and similar congeners exert a EDTA and DTPA) scavengers undergo a plausible electron
completely disparate effect on the peroxidase metabolism of transfer via the accessible lone pair of electrons of the
anilines from what has been described in this work for unprotonatedtertiarynitrogenatomtoformastabilizedradical
aminocarboxylates, and those amines, in fact, enhanced the cation that, at neutral pH, is rapidly decarboxylated, yielding a
generation of primary ANL radicals.3 In that same work, carbon-centered radical of the type −O CĊHNR (secondary
2 2
however, tetramethylethylenediamine, a tertiary amine with radical) with minimal formation of formaldehyde as a reaction
multiple methylresidues, led to theformation ofbothtypes of end product. This also implies that the intermediate reactive
free radicals (i.e., a mixture of N-centered and C-centered speciesinthiscaseisnotderivedfromtheattackatthecarbon
radicals in which the latter were the predominant species).3 bridge linking the nitrogen atoms. However, when mono-
Therefore, the triethylenetriamine backbone of DTPA is amincarboxylates (viz. MIDA and DMG) are the scavengers
partially responsible for scavenging ANL-derived primary N- used, although a similar initial electron transfer is proposed
centered radicals after enzymatic peroxidase activation. from the aromatic nitrogen to its aliphatic counterpart, the
The main conclusions of the EPR data are that (1) the ensuingshort-livedaminoacidderivativenitrogencationradical
coexistence of both the alkyl amine core of DTPA and the undergoes fast decarboxylation, yielding a putative carbon-
̇
carboxylic acid side chain are essential to mediate an electron centered radical of the type CH NR (primary radical). The
2 2
transfer from a tertiary amino group to ANL cation radicals latter readily gives off an electron (or electron equivalent) to
withthesubsequentformationofdaughterC-centeredradicals, atmosphericdioxygentogenerateaniminiumionwhichinturn
(2) the omission of the tertiary amine functionality abrogates hydrolyzes in aqueous solutions to yield ample amounts of
the free radical transfer from anilines, and (3) a tertiary rather formaldehydeinthereactionmedium(Scheme1).Overall,the
than a secondary nitrogen is indispensable for the attack of N- formation of a daughter secondary carbon-centered radical
centered ANL cation radical metabolites on the candidate (−R•CHR′−) by the peroxidative metabolism of arylamines
cosubstrate. Taking into consideration thermodynamic redox (e.g., aminoglutethemide) in the presence of a polyaminocar-
properties, tertiary amineswithoxidationpotentials wellbelow boxylate (e.g., DTPA) but not in the presence of a
their primary and secondary counterparts make them more monoaminocarboxylate (e.g., MIDA) was confirmed, at least
effective candidates to scavenge free radicals by direct electron in part, by the differential yield of CH O, which was
2
transfer.23,28 significantly lower in the case of the former (Figure 7).
Underaerobicconditions,scavengingofthecarbon-centered Nevertheless, this does not preclude the possibility of trapping
radical by molecular dioxygen at neutral pH results in the the initially formed primary carbon radical by DMPO in the
production of formaldehyde,23 the yield of which depends on case of polyaminocarboxylates before the rearrangement step
the type of the auxiliary free radical formed by charge transfer could take place. However, molecules that essentially lack
fromtheaniliniumcationradicalmetabolitestotheaminoacid available hydrogen atoms (e.g., MIDA and DMG) that could
molecule.Ifthefreeradicalgeneratedisaprimaryradical,then shift intramolecularly can produce only the primary radical
formaldehyde is a prominent reaction end product, whereas if species (i.e., −R•CH ) (Scheme 1). It is worth noting that the
2
the free radical generated undergoes rearrangement to a use of chromotropic acid to quantify formaldehyde was simple
secondary carbon radical, then formaldehyde is a minor and reliable and was validated by the fact that none of the
reaction end product. Interestingly, we showed indirectly that reactants or polymerization products absorb in the red region,
thefree-radicalintermediatethatformsthroughtheinteraction whereas the condensation of trace levels of formaldehyde with
ofaperoxidase-mediatedANLradicalmetaboliteandanamino chromotropicacidresultsintheformationofadimericadduct,
acid derivative differs by the type of the scavenger employed which, upon dehydration with a strong acid, yields the
1880 dx.doi.org/10.1021/tx4002463|Chem.Res.Toxicol.2013,26,1872−1883
Chemical Research in Toxicology Article
chromogen dibenzoxanthylium cation with a characteristic reactive carbon-centered radical intermediates. However,
purple color that absorbs at 580 nm.29 reduction of arylamine cation radicals by the scavenger
Kinetic spectrophotometry showed that the rate of cosubstrates can lead to higher levels of aryalmines available
peroxidase-mediated free-radical polymerization of ANL was for two-electron oxidation by the hepatocytes to arylamine
dramatically reduced when an aminocarboxylate molecule was electrophiles, which can lead to covalent binding of a different
added to the reaction mixture. In other words, we confirmed arrayofcellulartargets.Nevertheless,theone-andtwo-electron
that aminocarboxylate adjuncts (e.g., DTPA and MIDA) oxidation pathways are not mutually exclusive and could occur
suppress the generation of ANL polymers derived from N- simultaneouslyinthecaseofafirst-passeffectthroughtheliver
centered cation radical metabolites by interfering with the in addition to interactions with blood neutrophils and
latter, converting them to C-centered radicals that originate macrophages. Thebioavailability ofthearylamines inquestion,
from the adjunct molecules. Interestingly, the extent of the their volumes of distribution, their hepatic versus extrahepatic
suppression was significantly higher in case of amino acid clearances, and the immune and nutritional status of the
derivatives(eithermono-orpolyaminocarboxylicacidanalogs) individual might contribute to the level of toxicity posed by
than with DMPO given that identical volumes were added to these procarcinogens. The potential future biological applica-
the reaction mixtures and the same order of addition (prior to tions of the current work will aim at studying whether the
the arylamine substrate) was followed in each case (Figure 8). investigatedaminocarboxylatescavengerscouldconferacertain
This implies that the studied amino acid derivatives, which degree of redox protection in environmental toxicity involving
acted as chemical traps, are more potent than the spin trap arylamine xenobiotic exposure and human myeloperoxidase.33
■
DMPO in terms of scavenging ANL cation radicals. It is
noteworthy that decomposition or disproportionation radical CONCLUSIONS
and nonradical products as well as excess reactants (e.g.,
At physiological pH, a biochemical electron-transfer reaction
formaldehyde, CO , the aminocarboxylate ligand with one less
2
COOH group, and DMPO) do not absorb light at 450 nm.30 between amino mono- and polycarboxylic acids and anilinium
cation radicals derived from peroxidase-catalzyed oxidation of
As is well-established, AZO forms as one of the major
arylaminexenobioticsordrugsoccursinvitro.Weshowedalso
biotransformation products of ANL via oxidative free-radical
that the previous interaction is feasible in a whole cell lysate
coupling of two N-centered cation radical intermediates in
system. Polyaminocarboxylate metal chelators, such as DTPA,
biochemical reactions catalyzed by peroxidases, such as
HRP.31,32 We showed that DTPA, which behaved as a were transformed into oxygen-reactive carbon-centered radical
polyaminocarboxylic acid reductant, effectively and selectively intermediates by scavenging ANL-derived N-centered radical
metabolites. Simplification of the polyaminocarboxylate mole-
inhibited the peroxidase-dependent AZO production from
cule while maintaining some key functional groups, as in
ANL by diverting the self-dimerization reaction of anilinium
MIDA, a monoamino dicarboxylic acid, allowed an electron
cation radicals to the formation of a chemically new radical
transfer to and the subsequent reduction of nitrogen free
entity derived from the polyaminocarboxylate molecule.
radicalsoriginatingfromtheone-electronoxidationofANLby
Quantitatively, the one-electron oxidation of DTPA by an
horseradish peroxidase or HL-60 lysate in the presence of
equivalentamountofaniliniumradicalswasvirtuallycomplete,
catalytic amounts of H O . Likewise, ANL cation radicals
as determined indirectly by the total abrogation of azo-dimers 2 2
accepted an electron from DMG, an in vivo metabolite, that
that stem from parent N-centered radicals through the
was converted to the corresponding amino acid carbon-
peroxidative metabolism of ANL.
centered radical. In those radical-transfer reactions, it was
Althoughnitrogen-centeredcationradicalscanreacteitheras
possible, in terms of the differential yield of formaldehyde, to
electrophiles or free radicals by covalently binding to
determine tentatively the type of reactive intermediates
biomolecules or by abstracting a hydrogen atom from and
involved, whether a primary or a secondary carbon-centered
oxidizingkeycellularreductants(e.g.,GSH),they,comparedto
radical, depending on the type of scavenger. Compared to the
carbon-centeredradicals,areshorter-livedreactivespecies,have
spin trap DMPO, the studied amino acid derivatives are
lower redox potentials, are less reactive toward biological
macromolecules (e.g., DNA), and are oxygen nonreactive.1 apparently more potent scavengers of ANL-derived radical
Presumably, the biological prevalence of a specific metabolic metabolites because they both markedly reduced the rate of
peroxidase-triggered free-radical oligomerization and/or poly-
pathway, either the formation of nitrogen cation radicals and
merization of ANL and selectively inhibited the formation of
azo end products or the formation of carbon-centered free-
azobenzene, an ANL dimeric end product. The modulation of
radicalintermediatesandaldehydicendproducts,isanticipated
free-radical intermediates of the peroxidative metabolism of
to govern the ultimate potential cytotoxicity of arylamine
arylamines by amino acid derivatives will likely have
radical metabolites (Scheme 1). We envisage that the
toxicological consequences.
preferential reactivities of the reactive species corresponding ■
to the two metabolic pathways of arylamine xenobiotics/drugs
in the presence and absence of aminocarboxylates toward AUTHOR INFORMATION
protein targets contrasted with their reactivities toward cellular
Corresponding Author
antioxidants(e.g.,glutathione)isexpectedtodictatetheextent
*Tel.: [phone]. Fax: [phone]. E-mail:
of toxicity posed by each pathway in biological systems.
[email protected].
It is plausible that scavenging of nitrogen-centered aniline
radicals derived by one-electron oxidation of arylamine Funding
xenobioticsordrugsmightresultinattenuationoftheoxidative ThisworkwassupportedbyaresearchgranttoA.G.S.fromthe
stressand/or cytotoxicityattributedtotheseprocarcinogensin Canadian Institute of Health Research (CIHR reference no.
a promyelocyte cell system in spite of the fact that the 202034).K.M.istheholderofaclinicianfellowshipfundedby
aminocarboxylate scavengers produce the apparently more AIHS (201201086).
1881 dx.doi.org/10.1021/tx4002463|Chem.Res.Toxicol.2013,26,1872−1883