DTIC ADA539558: Free Radical Production from the Interaction of 2-Chloroethyl Vesicants (Mustard Gas) with Pyridine Nucleotide-Driven Flavoprotein Electron Transport Systems

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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas)

with pyridine nucleotide-driven flavoprotein electron transport systems 5b. GRANT NUMBER

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6. AUTHOR(S) 5d. PROJECT NUMBER
Brimfield, AA, Mancebo, AM, Mason, RP, Jiang, JJ, Siraki, AG, Novak, MJ,
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5f. WORK UNIT NUMBER

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT
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AUNSD A ArDmDyR MESeSd(iEcaSl) Research Institute of Aberdeen Proving Ground, MD
Chemical Defense 21010-5400 USAMRICD-P08-014
ATTN: MCMR-CDR-P
3100 Ricketts Point Road

9. SPONSORING / MONITORING AGENCY NAME(S ) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
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Published in Toxicology and Applied Pharmacology 234, 128–134, 2009.

14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Chemical warfare, Flavoenzyme, Chloroethyl mustards, Onium ion, Enzymatic reduction, Electron transport, Free radical, EPR
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OF ABSTRACT OF PAGES Alan A. Brimfield
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ToxicologyandAppliedPharmacology234(2009)128–134
ContentslistsavailableatScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas)
with pyridine nucleotide-driven flavoprotein electron transport systems☆
A.A. Brimfielda,⁎, A.M. Manceboa, R.P. Masonb, J.J. Jiangb, A.G. Sirakib,1, M.J. Novakc
aU.S.ArmyMedicalResearchInstituteofChemicalDefense,AberdeenProvingGround,MD,21010,USA
bLaboratoryofPharmacologyandChemistry,NIEHS,ResearchTrianglePark,NC,27709,USA
cDepartmentofChemistry,FloridaInstituteofTechnology,Melbourne,FL,32901,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Thebiochemicalsequelaetochloroethylmustardexposurecorrespondverywelltotoxicprocessesinitiated
Received15August2008 by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which
Revised30September2008 producecarbonfreeradicalswhenreducedelectrochemically.Therefore,wehypothesizedthattheonium
Accepted2October2008
ionsofsulfurornitrogenmustardsmightproducecarbonfreeradicalsuponbeingreducedenzymatically,
Availableonline15October2008
andthattheseradicalsmightconstituteametabolicactivation.Wesetouttodocumentradicalproduction
using an in vitro metabolic system and electronparamagnetic resonance (EPR). Our system consisted of
Keywords:
NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal
Chemicalwarfare
Flavoenzyme electron acceptor, various sulfur or nitrogen mustards and the spin trap α-[4-pyridyl-1-oxide]-N-tert-
Chloroethylmustards butylnitroneinbuffer.Reactionswerestartedbyaddingthereductasetotheothermaterials,vortexingand
Oniumion immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E
Enzymaticreduction EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a =15.483 G and
N
Electrontransport a =2.512 G. Theoutcomesupportedourhypothesisthatcarbon-centeredfreeradicalsareproducedwhen
H
Freeradical mustard-relatedoniumionsareenzymaticallyreduced.TheEPRresultsvariedlittlewiththechloroethyl
EPR
compoundusedorwithporcineorhumancytochromeP450reductase,thereductasedomainofratbrain
neuronalnitricoxidesynthaseorratliverthioredoxinreductase.Ourresultsoffernewinsightintothebasis
formustard-inducedvesicationandtheoutcomeofexposuretodifferentmustards.Thefreeradicalmodel
provides an explanation for similarities in the lesions arising from mustard exposure and energy-based
lesionssuchasthosefromheat,ultravioletandnuclearradiationaswellasdamageacrosstissuetypessuch
asskin,eyesorairwayepithelium.
PublishedbyElsevierInc.
Introduction species;noanimalblisterslikeman(Marshalletal.,1919).Recentin
vitrowork(Sabourinetal.,2000;Smithetal.,2001)hasrevealedthe
Sulfurmustard(2,2′-chloroethylsulfide[CAS505-60-2],mustard initiationofproinflammatorycytokineproductionatthecellularlevel
gas, NATO Standard Agreement designation; HD) and related startingseveralhoursafterexposure.However,theinitialbiochemical
chloroethyl compounds (Fig. 1) are alkylating agents and potent lesion, the event that ultimately sets off cytokine production and
vesicants.ItwaslastemployedincombatintheconflictbetweenIraq characteristicsymptoms,remainsunidentified.
andIraninthe1980s.Erythema,blistering,andnecrosisfollowdermal Severallinesofinquiryhaveindicatedthatthisinitiallesionmight
contact.Conjunctivitisand/orcornealopacityresultfromexposureof involve free radicals. For example, reports in the literature show a
the eyes. Inhalation can lead to pneumonia, chronic bronchitis and strongparallelbetweenthecellularandbiochemicaleffectsofsulfur
asthma,whilesystemicsymptomshaveradiomimeticcharacteristics. mustardexposureandtheeffectsoffreeradicaldamage(Papirmeister
Injury is dose dependent and initially painless (Papirmeister et al., etal.,1991;Somani,1992;HalliwellandGutteridge,1989).Addition-
1991;Somani,1992).Itisinterestingthatthedermalreactiontosulfur ally, when an electron rich center such as a sulfur, nitrogen or
mustard exposure in humans is different from that in most other seleniumisinavicinalrelationshiptoahalidesuchastheonefound
with these chloroethyl compounds (Fig.1), it favors intramolecular
☆ Theopinionsorassertionshereinaretheprivateviewsoftheauthorsandarenotto cyclization to form an energetically strained, reactive three-mem-
beconstruedasofficialorasreflectingtheviewsoftheArmyoroftheDepartmentof beredcycliconiumion(Fig.2)(Yangetal.,1988).Oniumions,cyclic
Defense. andacyclic,formspontaneouslyinsolutionsofmustards(Kangand
⁎ Correspondingauthor.Fax[phone].
Spears,1987;Ross,1962;Bartlettetal.,1949).Electrochemicalstudies
E-mailaddress:alan.a.brimfi[email protected](A.A.Brimfield).
1 Currentaddress.FacultyofPharmacyandPharmaceuticalSciences,Universityof haveshownthattheoneelectronreductionofoniumionsresultsin
Alberta,Edmonton,AlbertaCanadaT6G2N8. freeradicalproduction(SaevaandMorgan,1984).Reportsofthedirect
0041-008X/$–seefrontmatter.PublishedbyElsevierInc.
doi:10.1016/j.taap.2008.10.002
Author's personal copy
A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134 129
1991),thecytosolicthioredoxin-disulfidereductase[EC1.8.1.9](Gray
etal.,2007)andothers(HalliwellandGutteridge,1999)appeartoact
asgoodelectronsourcesforthisone-electronreductionprocess.
Ithasbeenknownforsometimethatoniumcompoundssuchas
diphenyliodonium ion and the dipyridylium ions of the herbicides
paraquat and diquat are capable of uncoupling extramitochondrial
electron transport and being reduced in place of cytochrome P450
or other terminal electron acceptors (Testa, 1995; Halliwell and
Gutteridge, 1999). Thediphenyliodoniumcationoffersanespecially
interesting example. When it undergoes single electron enzymatic
reduction by either cytochrome P450 reductase or the reductase
domain of nitric oxide synthase it rapidly fragments to yield
iodobenzeneandaphenylfreeradical(Tew,1993;Steuhretal.,1991)
capable of forming radical adducts (O’Donnell et al., 1994). This
providedadditionalevidencethatfreeradicalproductionmightbean
early step in the mechanism of mustard toxicity. As a result, we
hypothesized that a process mechanisticallyanalogous to that seen
with diphenyliodonium and paraquat led to the cytochrome P450
reductase(CYPOR)inhibitionthatwehadobservedandexplainedthe
parallel biochemical effects of free radical and mustard exposure.
Spontaneouslyformedoniumionscouldbeenzymaticallyreducedto
yieldcarbon-centeredfreeradicalswhichreactedwithanddamaged
cellularmacromoleculesleadingtotoxicity.
The detection of free radical formation from a mustard in the
Fig. 1. Compounds spontaneously forming “onium” ions. (A) Sulfur mustard, bis- presenceofNADPHandaflavoenzymereductaseinvitrowouldprove
(chloroethyl)sulfide(HD),(B)Chloroethylethylsulfide(CEES),(C)Chloroethylmethyl-
sulfide(CEMS),(D)Mechlorethamine(HN2),anitrogenmustard,(E)bis-(chloroethyl) the feasibility of this mechanism. Since an electron paramagnetic
selenide. resonance (EPR) signal is the preferred method for free radical
detection, we proceeded with the development of a spin trapping
technique able to detect carbon-centered free radicals emanating
electrochemical reduction of sulfonium ions in aqueous systems fromtheenzymaticoneelectronreductionofoniumionsoriginating
(Chambers,1978)indicatedthemechanisticfeasibilityofthisprocess withthemustards.
invivoandraisedthepossibilityofenzymaticreductionasthesource
ofmustardfreeradicals. Materialsandmethods
Additional evidence of free radical participation in mustard
toxicity arose as part of a study designed to evaluate the potential Reagents. RecombinantporcineCYPORandthereductasedomainof
forinteractionamongmilitarydeployment-relatedchemicalsatthe neuronalnitricoxidesynthase(nNOSR)fromratbrainwereakindgift
levelofmetabolism.Weinvestigatedtheeffectofsulfurmustardon fromProfessorBettieSueMastersoftheDepartmentofBiochemistry,
the microsomal cytochrome P450 drug metabolizing system using University of Texas Health Sciences Center at San Antonio.
NADP, an NADPH generating system and mouse liver microsomes Recombinant human cytochrome P450 reductase (HCYPOR) was
induced with phenobarbital. The results indicated that mustard purchased from BD Gentest, Bedford, MA. Rat liver thioredoxin
inhibitedtheO-demethylationofp-nitroanisolebycytochromeP450 reductase (TRXR), β-NADPH+H+, the spin trap α-(4-pyridyl-1-
(Brimfield and Hodgson, 2005). To clarify where mustard might be oxide)-N-t-butylnitrone (4-POBN), calmodulin from bovine brain,
exertingitseffect,wesimplifiedthemixturefromthepreviouswork cytochrome c from bovine heart,cantharidin, Chelex-100 and CaCl
2
by using purified NADPH–cytochrome P450 reductase (CYPOR) in werepurchasedfromSigmaChemicalCo.,St.Louis,MO.Thenitrogen
placeofmicrosomes,andcytochromecasaterminalelectronacceptor mustardmechlorethamine(methylbis(chloroethylamine),HN2),the
inplaceofthemicrosomalcytochromeP450.Employingthissystem monofunctionalsulfurmustardschloroethylethylsulfide(CEES)and
wewereabletofollowelectrontransportspectrophotometricallyby chloroethyl methyl sulfide (CEMS), the spin trap 2-methyl-2-
measuring cytochrome c reduction (Brimfield and Hodgson, 2005). nitrosopropane dimer (MNP) and trimethylsulfonium iodide were
The rate of cytochrome c reduction was slowed in the presence of purchasedfromAldrichChemicalCompany,Milwaukee,WI.Thealkyl
mustardjustastherateofp-nitroanisoleO-demethylationhadbeen. sulfonium ions triethylsulfonium iodide and triethylsulfonium
That effect provided an indication that the flavoprotein reductase
itself(theonlyremainingenzymeinthetestsystem)wastheprobable
siteofmustardinteraction.Thisphenomenonhasalsobeenreported
withhydrazineswhichareknowntoimpairtheoxidativemetabolism
of co-administered substrates in the microsomal drug metabolizing
systemandalsotobeenzymaticallyactivatedtocarbonfreeradicals
(Muakkassahetal.,1981;Reed,1976;LeeandLucier,1976).
Takentogetherthoseresultssuggestedthatmustardwasinhibit-
ingallcytochromeP450activitybyuncouplingelectrontransportat
the flavoenzyme reductase and diverting electrons directly to
xenobioticreduction.Thismodeoftoxicanttransformationcanlead
tofreeradicalformation(Testa,1995)andlendsweighttoourearlier
speculation about enzymatic onium ion reduction. Ubiquitous
pyridinenucleotide-drivenflavoenzymereductasessuchasNADPH– Fig. 2. The rapid Sn1 mediated internal cyclization of sulfur mustard with loss of
chlorinetoformthecyclicsulfoniumion.Thisschemeshowsnucleophilicattackby
cytochrome P450 reductase [EC1.6.2.4] (Tew, 1993), the reductase watertoformfirstthechlorohydrinandthen,aftertheformationofasecondoniumion
domainofneuronalnitricoxidesynthase[EC1.14.13.39](Steuhretal., andanotherreactionwithwater,thedoublyhydrolysedthiodiglycol.
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130 A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134
bromide were synthesized in the Department of Chemistry, Florida Table1
Institute of Technology, Melbourne, FL using methods described by Hyperfinesplittingconstantsresultingfromthespintrappingoffreeradicalsderived
fromflavoenzyme-reducedchloroethylmustardswith4-POBN
Lowe(1981).Sulfurmustard(98%purity)andtechnicalgrademustard
were obtained from the U.S. Army Edgewood Chemical Biological Enzyme Mustard Nitrogena Hydrogena
Center,AberdeenProvingGround,MD.Buffercomponentswerefrom aN(Gb) aH(G)
standard sources and were reagent grade or better. Water used in PorcinecytochromeP450reductase HD 15.46 2.54
conjunctionwithEPRstudieswasdeionizedto18.2MΩ/cmusinga CEMS 15.41 2.60
Milli-QPlusDeionizingSystem(MilliporeInc.,Billerica,MA). CEES 15.42 2.29
HN2 15.45 2.67
HumancytochromeP450reductase HD 15.40 2.68
The enzymatic spin trapping systems. Simple enzymatic assays CEMS 15.40 2.59
servedasthebasisforthedevelopmentofthespintrappingsystems CEES 15.43 2.27
andvariedsomewhatfromenzymetoenzyme.Theconcentrationof HN2 15.40 2.57
eachenzymewaskeptconstantafterweestablishedaworkableassay. Neuronalnitricoxidesynthasereductasedomain HD 15.40 2.69
Noeffortwasmadetorefinethesystemsfurther. CEMS 15.43 2.66
CEES 15.41 2.26
HN2 15.38 2.81
HCYPOR and CYPOR. The HCYPOR and CYPOR systems contained Thioredoxinreductase HD 15.42 2.70
0.016nMrecombinantenzymeasdescribedbyStrobelandDignam CEMS 15.41 2.61
CEES 15.50 2.13
(1978).Sulfurmustard(upto4.0mM),CEES,CEMSorHN2atdesired
HN2 15.54 2.63
mM levels and 2.4 mM NADPH were introduced with 40 µM
cytochromecin0.1MKPO bufferpH7.5made0.25Mwithrespect a Hyperfinesplittingconstantsfromcomputersimulationsoftheexperimentaldata
4 usingWin-Simsoftware.
toNaCl.Theconcentrationofthespintrap4-POBNwas1.03Mandthe b Gauss.
totalvolumewas500μL.
nNOSR. Theenzymaticsystemusedtodetectfreeradicalsformedby
ratbrainnNOSRwasmodeledonthatdescribedbyProfessorBettySue Electron paramagnetic resonance spectroscopy. With the exception
Masters (personal communication). The mixture contained 2.4 mM ofTRXR(seeabove),theEPRstudiesweredoneonaBrukerESP300E
NADPH,0.13µMenzyme,0.096µMcalmodulinand12.2mMcalcium EPRspectrometer(BrukerBiospin,Billerica,MA)equippedwitha4103
chloride.Itwasmadeupin0.05MtrispH7.4,containing0.1MNaCl,in TMcavityoperatingat9.80GHz.Theinvitrosystemsusedtoevaluate
place of the KPO buffer described above, to avoid precipitation of the reduction of sulfur and nitrogen mustards are described above.
4
calciumphosphate.Mustardconcentrationswereasmentionedabove. Thereactionwasstartedbyaddingthedesiredvolumeofasolutionof
The concentration of 4-POBN was 1.03 M and the total volume was thereductaseofinteresttoatubecontainingtheothermaterialsat
500μL. room temperature, vortexing and immediately transferring the
mixture to a 10 mm flat cell (Wilmad LabGlass, Buena, NJ). In the
TRXR. EPRstudiesusingTRXR(0.034μM)forenzymaticactivation interest of consistency, cytochrome c was included in the systems
wererunon a Bruker EMX Plus EPR spectrometerequippedwith a incorporating CYPOR,HCYPOR and nNOSRas a carry-over fromthe
4119HS cavity, and employed an enzyme system based on the one assay development stage in which the reaction was followed by
described by Elias et al. (1999). Briefly, the incubation mixture measuring the reduction of cytochrome c spectrophotometrically.
consisted of 2.4 mM NADPH,1.03 M 4-POBN, up to 4.0 mM sulfur CytochromecwasnotusedintheTRXRincubationmixture.Unless
mustardorconcentrationsoftheothermustardsnecessarytoyielda otherwiseindicated,theinstrumentsettingswere:microwavepower
freeradical signal,in Chelex-100treated 0.1 M KPO buffer, pH7.5. 10mW,modulationamplitude1.0Gauss(G),timeconstant327.68ms,
4
Totalvolumewas500μL. sweepwidth100G,centerfield3480Gandasweeptimeof1342.2s.
Eachspectrumwastheaccumulationofthreescans.Simulationswere
generatedusingWin-SimsoftwarecreatedatNIEHS.
Identification of the mustard free radicalstructure. Withtheuseof
thecorrectspintraponecandeducethestructureofaradicalfromthe
finesplittingpatternofitsspintrapadduct.However,because4-POBN
isanitrone,theadductformedoccurredatthecarbonadjacenttothe
imino nitrogen of the spin trap, too many bonds distant from the
protonsonthechloroethylgrouptoallowinteractionwithmagnetic
nuclei in the free radical to generate a useful hyperfine splitting
pattern(seeFig.7forstructures).
The nitroso spin trap MNP forms adducts at the nitrogenwhich
createsacloseenoughassociationbetweenthemagneticnucleiinthe
radical and the nitroso nitrogen of the MNP to provide the detail
neededtoallowdeductionoftheradical'sstructure.WeusedCEESin
placeofsulfurmustardforthestructuraldeterminationbecausethe
Fig.3.EPRspectrageneratedusingtheinvitrosystem.(A)Completeroomtemperature work was done at NIEHS rather than at the Institute of Chemical
incubationcontainingporcineNADPH–cytochromeP450reductase(0.016μM),2.4mM Defense.Inreality,thenon-sulfurmustardpartoftheradicalwastoo
NADPH,4.0mMsulfurmustard,1.03M4-POBNand0.21mMcytochromecin0.1M
remotefromthespintraptointerfere.Soitmadenodifferenceinthe
KPO4buffercontaining0.25MNaCl,pH7.5.(B)SameasinA,butwiththeporcine
reductasereplacedwithanequalvolumeofbuffer.(C)SameasA,butwithoutthe outcomewhetherweusedCEESorsulfurmustard.
NADPH.(D)SameasA,butwithoutsulfurmustard.Thesespectrawererecordedona MNP (3 mg) was dissolved in 1 mL of Chelex 100-treated
BrukerESP300Espectrometerequippedwitha4103TMcavityoperatingat9.80GHz, phosphate buffer (0.1 M, pH 7.4) overnight in a Thermomixer®
microwavepower:10mW, sweepwidth: 100G,midscale: approximately3488 G,
(Eppendorf, Germany), set at 30 °C and 1400 rpm. The MNP was
numberofscans:3,receivergain:1.25e5,timeconstant:327ms,conversiontime:
protected from light to prevent degradation. The complete reaction
655ms,modulationamplitude:1.0G.Allspectraarepresentedatthesamegain.The
10Gbarisincludedtodefinethescale. systemwaspreparedbyadding84mMCEES(neat,2μLfromstock)to
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A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134 131
Fig.4.Thedeterminationofthestructureofthemustardradical.TheexperimentalEPRspectrumofthecompletesystemforthedeterminationoftheradicalstructurecontaining
NADPH,CEES,HCYPORandMNPisshownin(A).(B)isthesameas(A)butwithouttheHCYPOR.Fig.4CcontainednoCEES.(D)containednoNADPHorHCYPOR.The10Gbaris
includedtodefinethescale.(SeeResults).
200 μL of the MNP solution which contained 3 mM NADPH. This soughtameanstodetectfreeradicalproductionwithoutambiguityby
mixturewasvortexedfor1min.Themixturewasmade64nMwith usingEPRspectrometrywithspintrapping.
respecttoHCYPORtoinitiatethereactionandwasthentransferredto Inclusionofthenitrone4-POBNinourincubationmixtureatthe
aquartzflatcellandmountedinthespectrometer. molar level enabled us to observe a six-line 4-POBN-radical adduct
Control experimentswerecarried out withoutHCYPOR,without spectrum when recombinant CYPOR was incubated with NADPH,
CEESorwithoutNADPHandthereductase.Thespectrawererecorded sulfurmustard,thespintrapandcytochromec(Fig.3A).Totestthe
withaBrukerEMXspectrometer(Billerica,MA)equippedwithanER proposition that each constituent was required for activity, we
4122SHQcavityoperatingat9.78GHzand100kHzmodulationfield repeated the EPR analysis in the absence of each component. As
atroomtemperaturewiththefollowingparameters:power=20mW, showninFigs.3B–Domittingreductase,NADPHormustardfromthe
scan rate=0.47 G/s, modulation amplitude=0.4 G, receiver otherwise complete in vitro mixture caused the production of free
gain=1.00×105. Each spectrum was the accumulation of four scans radicalstoceaseornearlycease,providingproofthateachcomponent
(355 s/scan, time constant=327 ms). Simulations were generated isrequiredforfreeradicalgeneration.
usingWin-SimsoftwarecreatedatNIEHS. Theinclusionofcytochromecintheincubationmixtureoriginated
withthecytochromecreductaseassayofStrobelandDignam(1978)
Molecular modeling. Electron density calculations for the deter- in which we first discovered the mustard–flavoenzyme interaction
mination of relative theoretical onium ion reducibility were per- and which subsequently served as a model for the free radical
formed with Spartan '04 software (Wavefunction, Inc., Irvine, CA) generatingsystem.Also,fromhisinvestigationofCYPORinhibitionby
usingB3LYPdensityfunctionaltheorywiththe6-31G⁎basissetand diphenyliodonium cation, Tew (1993) found that turnover was a
with geometryoptimization in the aqueous phase (Lee et al.,1988; prerequisite for inhibition of the reductase, and therefore, for free
Becke,1993). radicalgeneration.Wedidnotfindthattobethecasewiththeonium
species from the mustards. Running the enzymatic reaction using
Results NADPH, CYPOR and sulfur mustard, but neglecting to include
cytochromecinthemixturehadnoeffectonthequalityoftheEPR
Thetoxiceffectsproducedbysulfurmustardandthoseproduced signal produced (results not shown). In an unrelated experiment,
bytoxicprocessesknowntoactviafreeradicalgenerationaresimilar replacing mustard with the natural vesicant cantharidin in the
(Papirmeister et al., 1991; Somani, 1992; Halliwell and Gutteridge, incubation mixture (results not shown) produced no free radical
1989). Additionally, onium ions form spontaneously in solutions of signal. This may be an indication of an alternative mechanism for
mustard(KangandSpears,1987;Bartlettetal.,1949;Ross,1962)and cantharidin-inducedvesication.
reduction of onium ions leads to carbon-centered free radical
production (Chambers,1978, Stasko et al.,1993, Saeva and Morgan, DependenceofactivityononiumIonstructure
1984).Onthatbasiswehypothesizedthatmustardoniumionsmight
be enzymatically reduced by flavoenzyme reductases to produce Bothstrained,three-memberedcyclicsulfonium(Yangetal.,1988)
carbon-centeredfreeradicals.Inordertovalidatethathypothesiswe and immonium ions (Bartlett et al., 1949) as well as acyclic alkyl,
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132 A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134
Dependenceofactivityonreductase
RecombinantCYPOR,HCYPOR,ratbrainnNOSRandTRXRfromrat
livereachsupportedfreeradicalproductioninthepresenceofNADPH,
mustard,and4-POBN.Eachoftheseenzymesfunctionsaspartofan
extramitochondrial electron transport chain distributing reducing
equivalents from pyridine nucleotide coenzymes to metabolic end-
points.Eachpasseselectronstoaterminalelectronacceptoroneata
time via flavin prosthetic groups. The CYPOR, HCYPOR and nNOSR
accomplishthisbyemployingbothflavin-adeninedinucleotide(FAD)
andflavinmononucleotide(FMN)(Wangetal.,1997;Vasquez-Vivar
etal.,1999).IneachcaseFADacceptstwoelectronsfromNADPHand
transfersthemtoFMNwhichpassestheelectronsoneatatimetoa
terminal electron acceptor providing the sequential individual
electron transfers necessary for the reduction of cyclic onium ions
arisingfromchloroethylmustardsandtheproductionoffreeradicals.
TRXRisacytosolicenzymethatcatalyzestheNADPH-dependent
reduction of the active site disulfide in oxidized thioredoxin, a
powerful protein disulfide reductase catalyzing either electron
transport to ribonucleotide reductase and other reductive enzymes
orredoxregulationofenzymesandtranscriptionfactors.Itispartofa
mechanistically different family of enzymes, pyridine nucleotide–
disulfideoxidoreductases,havingonlyoneflavinprostheticgroup.In
thiscase,electronsarepassedfromNADPHviaFADtoanactivesite
disulfide,whichagainreducestheelectronacceptorthroughaone-
Fig. 5. Simulation of the experimental spectrum shown in Fig. 4C using Win-Sim
software.IntheabsenceofCEES,thespectruminFig.6Awasfound.Thehyperfine
splittingconstantsweresimulated,whichproducedthecloselycorrelatingspectrum
showninB.Onecomponentcontainingfoursharplines(Figs.5Dand6D)resultedfrom
thehydrogenfreeradicalcommonlyappearinginspintrappingexperimentsusing
MNP. The other component containing six lines only appeared in the absence of
CEES and is thought to arise from the formation of a carbon-centered radical
present in the enzyme preparation. The 10 G bar is included to define the scale.
(SeeResults).
unstrainedcyclicandpolymericoniumions(St.Quintinetal.,2003;
Blacketal.,1992)forminmustardsolutions.Wedidnotdifferentiate
among these in our original hypothesis regarding onium ion
reduction.We feltthatfreeradicalswouldbeformedbyenzymatic
reductionofoniumionsregardlessoftheirstructure.
However, free radicals were only detected when sulfur mustard
(Fig.1A), CEES (Fig.1B), CEMS (Fig.1C), HN2 (Fig.1D) or technical
sulfurmustard(acomplexmixtureofmustardandrelatedmaterial
not shown) were included in the reaction mixture. Our original
hypothesispredictedfreeradicalformationbyenzymaticreductionof
all onium ions. To test this idea, we tested stable, non-cyclic alkyl
onium ions such as trimethylsulfonium iodide, triethylsulfonium
iodideandtriethylsulfoniumbromide.Noneofthecompoundslacking
thechloroethyl groupand incapable offormingthestrained,three-
memberedcycliconiumionsgaverisetoafreeradicalsignalbyEPR
usingourinvitrosystem(datanotshown).
Inanattempttoexplainthisdifferenceinenzymaticreducibility
weapplieddensityfunctionaltheorymodeling(Leeetal.,1988;Becke,
1993). This technique allowed us to evaluate molecular features to
provideatheoreticalframeworkbywhichtointerpretthevariability
in our results arising from ion structure. The calculations for
sulfoniumionsinanaqueousenvironmentindicatedthattheenergy
of the lowest unoccupied molecular orbital (LUMO) in the strained
ring systemwas lower (−0.93 eV) than the energy of the LUMO of
acyclicalkylsulfoniumions(−0.81eV).Intheory,atleast,thismeans
the cyclic ions can acceptan electron more readily than the acyclic Fig.6.SimulationoftheexperimentalspectrashowninFig.4usingWin-Simsoftware.
oniumionscan.Therefore,anenzymaticsystemhavingalessrobust (A)istheoriginalexperimentalspectrumofthespintrappedMNP-CEESfreeradical
reduction potential than direct electrochemical reduction or a adduct.(B)isthecomputersimulationof6A.(CandD)showtheindividualcomponents
ofthesimulation.(C)istheMNPtrappedfreeradicalgeneratedbytheenzymatic
chemical reducing agent is capable of donating an electron to the
reductionofCEES.(D)isthetrappedhydrogenfreeradicalcommonlyappearingin
strainedcyclicthree-memberedringandlesscapableofreducingthe
controlandspintrappingexperimentsemployingMNP.(D)contributes15.6%ofthe
morestablealkyloniumions. areaunderthetotalspectrumshownin(B).The10Gbarisincludedtodefinethescale.
Author's personal copy
A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134 133
electrontransfer(MustacichandPowis,2000).Theendresultisthe
same.Thereareadditionalreductasesinthisenzymefamilythatmay
alsobecapableofreducingthecycliconiumions.
Table1showsthehyperfinesplittingconstantscalculatedfromthe
computer-basedsimulationsofthespintrapadductspectraresulting
fromthereductionofeachofthechloroethylvesicantsbyeachofthe
four enzymes tested. The data given for the reduction of sulfur
mustardbyCYPORarefromthespectrumshowninFig.3A.Alongwith
thegenerationofadetectableEPRsignalbyeachofthereductases,the
similarityamongthehyperfinesplittingconstantsfromthespectraof
the4-POBNadductssuggestsacommonalityofoutcomesarisingfrom
the reduction carried out by the different enzymes. The process
illustrated in Fig. 7 lends weight to this conclusion as well. These
results provide a bridge between the outcome of chemical and
electrochemical reduction of onium ions leading to free radical
production(SaevaandMorgan,1984;Chambers,1978)andwhatwe
seewithourinvitroenzymaticsystem.
Identityofthefreeradicalstructure
Determinationofthefreeradicalstructurewasdonebygenerating
theadductwiththespintrapMNPusingCEES(Fig.1B)ratherthan
sulfur mustard as the source of the radical in the in vitro enzyme
system. CEES provided an EPR spectrum qualitatively equivalent to
thatofsulfurmustardwhenweusedourinvitrosystemwithHCYPOR
asthereductaseand4-POBNasthespintrap(Fig.3A)(Brimfieldand Fig. 7. The proposed pathwayfor the production of free radicals from chloroethyl
Mancebo,2007).Therefore,wehadnoreasontoexpectanydifference mustards.Fig.7givesthestructureofthefreeradicalsformedfromtheinteractionof
theflavoenzymereductaseswithchloroethylmustards,andthestructureofthespin
arisingfromtheuseofCEESinplaceofsulfurmustardwithMNP.
adducts formed with the spin traps 4-POBN or MNP. The compound shown is
TheEPRspectrumofthecompletesystemisshowninFig.4A,Figs.4B chloroethylethylsulfide.However,theprocesswouldbethesamewithanyof the
toDarethecontrolexperiments.Fig.4Cshowsthesignalgenerated chloroethylmustardsincludingsulfurmustard.
withoutCEES,andFig.4DshowstheeffectofleavingoutNADPHand
HCYPOR.WhenHCYPORwasleftoutofthemixture(Fig.4B)thefree
radicalsignalwassignificantlylower. also seen in Fig. 5D and commonly appearing in control and spin
WithoutCEES(Fig.4C)thespectrumshowedtwocomponentsof trapping experiments employing MNP (Kalyanaraman et al., 1979;
backgroundsignal.One(indicatedby⁎)wasidentifiedbycomputer Mottley et al., 1981), where a =14.62 G and a =13.95 G. The
U N H
simulation (Fig. 5D) as the MNP/H radical commonly appearing in hydrogenradicalcontributes15.6%ofthewholespectrum.
control and spin trapping experiments employing MNP (Kalyanara-
manetal.,1979;Mottleyetal.,1981),whiletheotherspecieswiththe Discussion
U
six-linespectrum(indicatedby )wasnotdetectedinthecomplete
system. Simulation of the six-line spectrum (Fig. 5C) provided Untilnowinjuryfromexposuretomustardandrelatedvesicants
hyperfine splitting constants a =3.65 G and a =15.8 G with a has been assumed to result from direct alkylation of biological
H N
correlation factor of 0.97. It is likely that this signal was derived macromoleculesbycycliconiumions.Theresultsofthisinvestigation
U
from a carbon-centered radical with one hydrogen (CH(R) ) — offer an additional mechanism by which to explain the outcome of
2
possibly a component present in the enzyme preparation. (Note: mustardexposureofskin,eyeandairwayepitheliumintheformoffree
thiscontrolexperimentusedadoublingofthetimeconstantanda radical-relatedalkylation.Itisnotnecessarytoinvokeoneortheother
doublingof the conversion time toreduce the noise. The scalewas ofthesetoxicologicprocessesexclusively.Bothdirectalkylationand
adjustedtobeequivalenttotheotherspectra.) radicaldrivenalkylationcouldbeatworksimultaneously.Following
TheexperimentalEPRspectrainFig.4weresimulatedusingWin- the formation of the cyclic sulfonium ions, we cannot exclude the
Simsoftware(Fig.6).Fig.6Aistheexperimentalspectrumproduced possibilityofanenzymaticreduction/freeradicalmechanismactingin
bythecompletesystem.Fig.6BisthecomputersimulationofFig.6A. parallelwiththetraditionallyassumeddirectalkylation.Onecaneven
Acorrelationfactorof0.96indicatedthatthesimulationwasexcellent. view flavoenzyme reduction and direct electrophilic alkylation as
Figs. 6C and D show the two components of the 6B simulation processescompetingfortheavailablecyclicsulfoniumionsupply.This
separately.Fig.6CistheMNPtrappedfreeradicaladductgenerated isaveryinterestingperspective.Intheeventofreductaseinhibition,
using CEES. The hyperfine structure includes a single nitrogen free radical generationwould cease, and the available cyclic onium
splitting of a =16.63 G, splitting of two equivalent hydrogens specieswouldformmacromolecularadductsviaelectrophilicalkyla-
N
aβ=11.24 G representing interaction with the hydrogens on the α tion.Inthatcasesignsoftoxicitywouldcontinuetoaccumulatefrom
H
carbon and another splitting of two equivalent hydrogens aγ=0.56 non-freeradical-relatedalkylationoriginatingwiththecyclicspecies.
H
representing the effect of the hydrogens on the β carbon of the However,iftoxicitywereterminatedbyreductaseinhibitionitwould
chloroethylgroup.TheCEESradicaladductcontributes84.4%ofthe pointtofreeradicalgenerationasthesolesourceofmustard-related
entireareaunderthecurve.Theproposedreactionandthestructure tissuedamageandprovideanadditionaldirectiontoguideoursearch
of the free radical generated are shown in Fig. 7. The hyperfine fortherapeuticstrategies.
splittingsfromthesimulationarewhatwouldbeexpectedfromthe Wecancarrythislineofreasoningafewstepsfurther.Ourresults
free radical trapped by MNP in Fig. 7, which features a nitrogen showing enzymatic reduction of cyclic onium ions support work
splitting, and splitting by two sets of two equivalent protons reportedseveralyearsagoinaseriesofpapersbySawyer(1998,1999;
contributed by the CEES radical (Makino, 1979). Fig. 6D is the Sawyeretal.,1996)outlininghisinvestigationofnitricoxidesynthase
U
simulation of the spectrum for the hydrogen free radical (MNP/H) (NOS)involvementinsulfurmustardinjury.Usingprimaryculturesof
Author's personal copy
134 A.A.Brimfieldetal./ToxicologyandAppliedPharmacology234(2009)128–134
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