DTIC ADA539503: Evaluation of Protease Inhibitors and an Antioxidant for Treatment of Sulfur Mustard-Induced Toxic Lung Injury

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Evaluation of protease inhibitors and an antioxidant for treatment of sulfur mustard-induced

toxic lung injury 5b. GRANT NUMBER

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Anderson, DR, Taylor, SL, Fetterer, DP, Holmes, WW
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AUNSD A ArDmDyR MESeSd(iEcaSl) Research Institute of Aberdeen Proving Ground, MD
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Published in Toxicology, 263, 41–46, 2009
14. ABSTRACT
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15. SUBJECT TERMS
Sulfur mustard, Lung injury, Pulmonary function, Treatment
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Toxicology263(2009)41–46
ContentslistsavailableatScienceDirect
Toxicology
journal homepage: www.elsevier.com/locate/toxicol
Evaluation of protease inhibitors and an antioxidant for treatment of sulfur
mustard-induced toxic lung injury
DanaR.Andersona,∗,StephanieL.Taylorb,DavidP.Fetterera,WesleyW.Holmesa
aMedicalToxicologyBranch,AnalyticalToxicologyDivision,U.S.ArmyMedicalResearchInstituteofChemicalDefense,3100RickettsPointRoad,
AberdeenProvingGround,MD21010,USA
bUniversityofDelawareSchoolofNursing,McDowellHall,25NorthCollegeAvenue,Newark,DE19716,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Sulfurmustard(SM)-inducedlunginjuryhasbeenassociatedwithproteaseactivation,oxidativeinjury
Received1April2008 andinflammatoryresponseculminatingintissuenecrosis.Theproteaseinhibitorsaprotininandilo-
Receivedinrevisedform25July2008 mastatandtheantioxidanttroloxwereevaluatedforefficacyinamelioratingSM-inducedlunginjury.
Accepted19August2008
Anesthetizedspontaneouslybreathingrats(N=6–8/group)wereintratracheallyintubatedandexposed
Availableonline21September2008
to1.4mg/kgSM(0.35mgSMin0.1mlofethanol)orethanolalonebyvaporinhalationfor50min.At
1minbeforetheexposureratsweretreatedwithoneofthefollowing:intravenousaprotinin,4.4mg/kg;
Keywords: intraperitoneal(ip)ilomastat,25mg/kg;oriptrolox,500(cid:2)g/kg.Aprotinin-treatedanimalsreceivedsup-
Sulfurmustard
plemental2.2mg/kgdosesat1minand6hpost-exposure(PE).Awholebodyplethysmographsystemwas
Lunginjury
Pulmonaryfunction usedtomonitorpulmonaryfunction(PF)parametersfor1hbeforeexposure(baseline),andfrom5–6and
Treatment 23–24hpost-exposure.SMinhalationcausedsignificantincreasesinseveralPFparameters,includingtidal
volume,peakinspiratoryflow,peakexpiratoryflow,endexpiratorypauseandenhancedpause.Consistent
withthereporteddevelopmentofSM-inducedpathology,thesechangeswereminimalatthe5–6-htime
andsignificantatthe23–24-htimepoint.Atthelatertimeitisknownfrompreviousworkthatairways
arebecomingobstructedwithloosecellulardebris,damagedcellsandexudate,whichcontributedtothe
changesinPFparameters.TreatmentwithaprotininorilomastateliminatedthesePFchanges,yielding
resultscomparablewithcontrolsforeachoftheseparameters.LunglavagefluidanalysisshowedthatSM
causedasignificantincreaseintotalprotein(TP)andinthecytokinesIL-1(cid:3)andIL-13.Aprotinintreat-
mentpreventedtheincreasesinTPandIL-1(cid:3)production,ilomastatpreventedtheincreasedproduction
ofIL-13,andtroloxtreatmentdidnotsignificantlypreventtheSM-relatedincreasesinTP,IL-1(cid:3)orIL-13.
Histopathologicexaminationoflungtissue24hpost-exposureshowedminimalalveolareffectscausedby
SM,whiledamagetobronchiolarregionswasmuchmoresevereduetothehighlyreactivenatureofSM.
WhileaprotininandilomastatbothalleviatedthePFperturbations,surprisinglyonlyaprotininreduced
theobservedpathology,bothgrosslyandhistologically.Theseearlyresultsindicatethattreatmentwith
aprotininandtoalesserextentilomastatreducessomeofthedirectinflammatoryresponseanddamage
associatedwithSM-inducedlunginjury.
ThisresearchwassupportedbytheDefenseThreatReductionAgency–JointScienceandTechnology
Office,MedicalS&TDivision.
PublishedbyElsevierIrelandLtd.
1. Introduction individualincapacitatedfordaystomonths,dependingonthedose.
This not only removes the individual from vital duties, but also
Sulfurmustard(2,2-dichlorodiethylsulfide,SM)isachemical placesastrainonthemedicalsystem.Duetotheavailability,ease
warfareagentthatremainsathreattothetacticalstrategiesofthe ofproductionandstorage,persistenceandstability,SMisconsid-
United States Army and the health of the US soldier. More than eredapotentialterroristagentthatposesathreattothecivilian
lethality,SMcausesdebilitatingeffectsthatcanleaveanexposed populationaswell.Despitedecadesofresearch,nocompletelysat-
isfactoryexplanationofthetoxicactionofSMexists,andasaresult,
the development of an effective antidote has not been realized
(Papirmeisteretal.,1991).
∗
Correspondingauthor.Tel.:[phone]. Respiratory damage resulting from SM inhalation exposure is
E-mailaddresses:[email protected](D.R.Anderson),
dose-dependent and latent in onset. Inhalation of low to mod-
[email protected](S.L.Taylor),[email protected](D.P.Fetterer),
[email protected](W.W.Holmes). erate levels of SM primarily affects the upper respiratory tract
0300-483X/$–seefrontmatter.PublishedbyElsevierIrelandLtd.
doi:10.1016/j.tox.2008.08.025
42 D.R.Andersonetal./Toxicology263(2009)41–46
with alveolar damage occurring only with high dose exposures 2. Methods
(Papirmeisteretal.,1991).Insevereexposures,SMmayenterthe
circulatorysystemandaffectotherorgansystems,suchasthebone 2.1. Animals
marrow and lymphatic tissue. The associated suppression of the
Malerats(Crl:CDSDBR),weighing230–260g,werepurchasedfromCharles
immunesystemleavesanexposedindividualsusceptibletoinfec- RiverLaboratoryandmaintainedinananimalcarefacilitythatisfullyaccreditedby
tion.ThecombinedeffectofSM-inducedrespiratorylesionsand theAssociationforAssessmentandAccreditationofLaboratoryAnimalCareInterna-
infections secondary to SM-related immunosuppression such as tional.Inconductingtheresearchdescribedinthisreport,theinvestigatorsadhered
tothe“GuideforCareandUseofLaboratoryAnimals,”revised1996.
bronchopneumonia has been documented as the leading cause
ofmortalityinWWI(Papirmeisteretal.,1991)andtheIran/Iraq
2.2. Intubation&SMexposure
war(Willems,1989).Long-termeffectsofSMinhalationexposure
include the development of chronic obstructive pulmonary dis- Thisexposuremodelhaspreviouslybeendescribed,andtheSMdoseemployed
ease(mustardlung),bronchiectasis,asthma,andfibrosis(Emadand hasbeenshowntoproduceconsistent,non-lethalpathologyat24hpost-exposure
(Andersonetal.,1996).SMwasdilutedto3.5mgml−1inabsoluteethanol.Ratswere
Rezaian,1997).
anesthetizedwithanintramuscularly(im)administeredcombinationofketamine
Although not fully understood, SM-induced cytotoxicity is (80mg/kg)andxylazine(10mg/kg),andintubatedwithamodifiedglassPasteur
attributedtoitsalkylatingproperties.Thelipophiliccharacteristics pipette(ca.5cmlong)usingalaryngoscopetovisualizethelarynxandapieceof
ofSMallowittoreadilycrosscellularmembranesandreactwith PE90tubingasaguidetube.Thelengthoftheglassendotrachealtubewassufficient
fordistalplacementinthetracheaatthepointofthelarynxandthebifurcationof
manybiologicallyimportantmolecules,suchasDNA,RNA,proteins
thetrachea.Onceinplacetheendotrachealtubewassecuredbywrappingapiece
andothermembranecomponents.ThedownstreameffectsofSM-
ofporoustapearoundthetubeandtherostrumoftherat.Aglassendotracheal
inducedDNAalkylationhavebeenpostulatedtoincludeanincrease tubewasnecessarytominimizeabsorptionofSM.100(cid:2)lofSM(0.35mg)inabso-
inproteaseinductionandactivation(Papirmeisteretal.,1991).Sev- luteethanolorethanolalone(control)wasplacedinawaterjacketed(37◦C)glass
eralinvestigatorshaveprovidedsupportingevidencefortherolefor vaporgenerator(customfabricatedbyAtmarGlass,KennettSquare,PA),andspon-
taneouslybreathingratswereconnectedtothisdeviceandexposedfor50min.
proteasesinthepathogenesisofSM-induceddamage.Thesestud-
Bytheendoftheexposureperiod,theSMinethanolwascompletelyvaporized
iescoverawiderangeofproteaseclassesincludingserine,cysteine, andinhaled.Thispassiveexposuresystemincludesaninletone-wayrespiratory
andmatrixmetalloproteases(Woessneretal.,1990;Cowanetal., checkvalve(HansRudolph,Inc.,KansasCity,MO)toinsurethattheonlysourceof
1991,1997;Casillasetal.,2000;Guignabertetal.,2005;Shakarjian airfortheanimalduringtheexposurewasthroughthevaporgenerator.Exhaled
airpassesthroughatwo-waynon-rebreathingRudolphvalveandthenthrougha
etal.,2006).Increasedproteolyticactivityhasbeenreportedwith
charcoal-filteredbleachtraptodecontaminateanyexhaledSM.Attheconclusion
theexposuremodelusedherein(Cowanetal.,1997).
ofthe50-minexposure,theratsweredisconnectedfromthevaporgenerator,the
InadditiontoDNAalklyation,SMmayreactwithanddeplete endotrachealtubewasremoved,andtheratswerereturnedtotheircage.
reducedglutathione(GSH)levels.GSHisamoleculethatisinvolved
in the detoxification of endogenous and exogenous sources of 2.3. Treatment
reduced oxygen radicals (Papirmeister et al., 1991). Free oxygen
Ratswereassignedtooneoffivetreatmentgroups(N=6–8):ethanolexposed/no
radicals react with membrane phospholipids, creating lipid per-
treatment (control); SM-exposed/no treatment; SM-exposed/aprotinin-treated;
oxides that attack adjacent lipids. This chain reaction can lead SM-exposed/ilomastat-treated; SM-exposed/trolox-treated. 1min prior to SM
to alterations in membrane fluidity, loss of membrane protein exposureratsweretreatedwithaprotinin(SIGMA,St.Louis,MO),4.4mg/kg,intra-
function, disruption of membrane integrity and ultimately cell venously(iv);ilomastat(QuickMedTechnologies,Inc.,Gainesville,FL),25mg/kg,
intraperitoneally(ip);ortrolox(SIGMA,St.Louis,MO),500(cid:2)g/kg,ip.Tomaintaina
death. Anderson et al. (1993) reported that SM may affect lungs
sufficientdoseofcirculatingaprotinin(half-life0.7h;Levyetal.,1994)ratsassigned
byfreeradical-mediatedmembraneandtissuedamage.Thisstudy tothistreatmentgroupwereadministeredadditional2.2mg/kg,ivinjectionsat
identifiedthegenerationofanascorbylradicalat1and24hpost- 1minand6hPE.Thetotalaprotinindose(8.8mg=∼80,000KIU)iswithintheclini-
exposure (PE) and thiobarbituric acid reactive products (TBARs) callyrelevantdoserangeof60,000–120,000KIU/kg,andwasusedbyAsimakopoulos
etal.(2000),asananti-inflammatorydoseinrats.Thedoseofilomastatwasrecom-
24hPEinbronchoalveolarlavagefluid(BALF),whichindicatedthat
mendedtobyDr.RichardGalardyfromQuickMedTechnologies,asadosetousewith
lipidperoxidationandmembranedamagehadoccurredinresponse
asystemicinjectioninrats.Duetotheslowclearanceofilomastat(half-life5–6h),
toSMinhalation. wefeltonedosewasadequateforthesepreliminarystudies.Thedosefortroloxwas
Epithelial and other cell type damage seen in SM toxicity is fromKumaretal.(2001),whoreportedincreasedsurvivaltimesusingtroloxagainst
associatedwiththereleaseofextracellularproteasesandoxidative sulfurmustardinhalationinjury.Intheirstudytroloxwasgivenoncein24h,there-
forewefollowedthissameschedule.Alldrugswerepreparedinphosphatebuffered
radicalsthatareresponsibleforthemajorityofallinflammatory,
saline.
coagulative,andtissueremodelingprocessespluscellularmem-
branedestruction(Cowanetal.,1997;PutnamandRoyston,2003; 2.4. Wholebodyplethysmography(WBP)
Papirmeisteretal.,1991).Undernormalornon-diseasedphysio-
logical states there is a balance between protease inhibitors and A Buxco unrestrained WBP system with Biosystems XA software (Buxco
ResearchSystems,Wilmington,NC)wasusedtomonitorpulmonaryfunction(PF)
proteasesaswellastheoxidizedandreducedmolecules.However,
parametersinconsciousrats.Theseparametersincludedtidalvolume,peakinspi-
inacutetissueinjurythesebalancescanbeoffset,resultinginan ratoryflow,peakexpiratoryflow,endexpiratorypause,andenhancedpause,an
environmentdominatedbyproteasesandfreeradicals.Moreover, indirectmeasureofbronchoconstriction.Priortoethanol(vehiclecontrols)orSM
Guignabertetal.(2005)showedthatlevelsoftissueinhibitorsof exposure,ratswereplacedintheWBPchambersforatotalof2h(1hforaccli-
mationand1hfordatacollection)toestablishabaselineforPFparameters.At
metalloproteases(TIMPS)werenotsufficienttorepressincreased
theconclusionofeachSMexposure,theratswereplacedintheiroriginalcages
gelatinase activity in SM-induced respiratory lesions in guinea torecoverfromanesthesia.FourhourPEtheratswereplacedintheWBPcham-
pigs. bersagainandallowedtoacclimatefor1h;PFdatawasrecordedfrom5to6h
Proteaseinhibitorsandfreeradicalscavengershavebeenprevi- post-exposure.Thefollowingday,PFparameterswereassessedfor1haspreviously
ouslysuggestedandtestedaspotentialtreatmentsforSM-induced described,throughthe24-htimepoint.Datawereaveragedbygroupforeach1-h
datacollectionperiod.
injury with some success in both in vitro and in vivo models
(Lindsayetal.,1996;Cowanetal.,1997,2002;Guignabertetal., 2.5. Bronchoalveolarlavage(BAL)
2005).Therefore,theaimofthisstudywastofurthertesttheeffi-
cacyofsupplementaladministrationofotherproteaseinhibitors Twenty-fourhourPE,ratsweredeeplyanesthetized,abloodsamplewasdrawn
fromthedorsalvenacava,andthentheratswereexsanguinatedbyclippingthevena
andanantioxidantcompound,includingthebroadspectrumpro-
cavaanddescendingaorta.Differentialbloodcellcountswereperformedusinga
teaseinhibitorsaprotininandilomastatandtheantioxidanttrolox
CELL-DYN3500(AbbottLaboratories,AbbottPark,IL).Thetracheawasisolated,
againstSM-inducedpulmonarytoxicity. anda16ga.gavageneedlewasintroducedintothetracheaandsecuredusing3/0
D.R.Andersonetal./Toxicology263(2009)41–46 43
suture.Thechestcavitywasopenedandthetrachea/heart/lungblockwasremoved. thesection.Incidenceofpathologywasnotedforthefourrightlungsectionsand
PE50tubingwaspassedthroughthegavageneedleandtiedintotheleftmainstem thenrecordedassectionsshowingpathologyversustotalnumberofsections.
bronchustoserveasacatheterforbronchoalveolarlavage.Theleftlungwaslavaged
threetimeswith3-mlaliquotsofsaline,whichwascollectedvialow-pressurevac- 2.7. Biochemicalevaluation
uum.Theleftlungwastiedoffandremovedfollowinglavage.TherecoveredBALF
wasimmediatelypooledandcentrifuged(10minat300×g),andthesupernatant BALFsupernatantwasanalyzedfortotalprotein(TP),IL-1(cid:3),andIL-13.TPlev-
wasremovedandstoredat−80◦Cuntilbiochemicalanalyseswereperformed. elsweredeterminedusingaBio-RadProteinAssay(Hercules,CA)modifiedfora
96-wellplate.Absorbancewasreadat595nmusingaSPECTRAmaxPlus384(Molec-
2.6. Histopathology ularDevices,Sunnyvale,CA),microplatespectrophotometer.Theconcentrationsof
cytokinesIL-1(cid:3)andIL-13weredeterminedusingaLinco-plexmulti-plexedcytokine
Immediatelyfollowinglavage,thetracheaandthefourrightlunglobeswere assay(Linco,St.Charles,Missouri)andanalyzedonaBio-Plexmultiplexanalyzer
removedandformalinfixed(10%bufferedphosphateformalin,FisherScientific, (Bio-Rad,Hercules,CA).
Pittsburgh,PA)forhematoxylinandeosinstainingandevaluation.Allrightlung
lobes(cranial,medial,caudal,andaccessory)wereassessedforextentofdamage 2.8. Dataanalysis
basedonthefollowingparameters:bronchialexudate,bronchialepithelialnecro-
sis,bronchialpolymorphonuclear(PMN)infiltrates,bronchialassociatedlymphoid Thedataareshownmean±S.E.forbothtextandfigures.Groupcomparisons
tissue(BALT)necrosis,alveolarexudate,alveolarhemorrhage,alveolarPMNinfil- wereconductedusingone-wayANOVAfollowedbyTukey’spost-hocmultiplecom-
trates,alveolarepithelialnecrosis,andperivascularfibrin/edema.Eachparameter parisontest.PathologydatawereanalyzedacrossgroupswithaChisquaretest.If
wasgivenascoreof0through3:0=normal;1=minimal,presentin1–10%ofthe itwassignificant,allgroupswerecomparedtoEtOHandalsotoHDaloneusinga
section;2=moderate,presentin10–50%;3=severe,presentingreaterthan50%of Fisher’sexacttest.Significantresultswereidentifiedwhenp<0.05.
Fig.1. PulmonaryfunctionparametersmeasuredontheBuxcowholebodyplethysmographysystem.SMinhalationcausedsignificantincreases(*)relativetocontrol(EtOH)
inseveralparametersat24hpost-exposure,includingtidalvolume(A),peakinspiratoryflow(B),peakexpiratoryflow(C),endexpiratorypause(D)andenhancedpause(E).
Treatmentwithaprotinin,ilomastatandtoalesserextenttroloxamelioratedtheseeffects.
44 D.R.Andersonetal./Toxicology263(2009)41–46
3. Results
3.1. Pulmonaryfunctionparameters
SMinhalationcausedsignificantincreasesinseveralparame-
ters,includingtidalvolume,peakinspiratoryflow,peakexpiratory
flow,endexpiratorypauseandenhancedpause(Fig.1A–E).Con-
sistentwiththereporteddevelopmentofSM-inducedpathology,
these changes were minimal at the 5- to 6-h timepoint and sig-
nificant at the 23- to 24-h timepoint. It is known from previous
work (Anderson et al., 1996) that by 24h, airways are becoming
obstructedwithloosecellulardebris,damagedcellsandexudate.
ThesephysicalobstructionsmaycontributetothechangesinPF
parametersobservedhere.Increasesintheseparametersarecon-
sistentwiththedeepgaspingbreathsoftheSM-exposedanimals
havingtoworkhardertobreaththroughpartiallyoccludedairways.
Treatmentwithaprotininorilomastateliminatedthesechanges,
yieldingresultscomparablewithethanolcontrolsforeachofthese
parameters.Theantioxidanttroloxgenerallywasnotaseffectiveas
thetwoproteaseinhibitors,reducingonlytheSMeffectontidalvol-
ume(Fig.1A),endexpiratorypause(Fig.1D)andenhancedpause
(Penh,Fig.1E).
3.2. Biochemicalevaluation
We,aswellasothers,haveusedBALFtocharacterizethetempo-
ralbiochemicalchangeswithinthebronchoalveolarregionofthe
Fig.2. Biochemicalassaysonlunglavagefluid.Totalprotein(TP)(A);interleukin1
lungfollowingaSMexposure,andthesechangesarequantifiedto
alpha(IL-1(cid:3))(B);andInterleukin13(IL-13)(C).N=6–8pergroup.(*)Significantly
determinetheefficacyoftestedtherapeuticcompounds(Anderson
differentfromEtOH.(**)SignificantlydifferentfromEtOHandSM.
etal.,1997;Cowanetal.,1997;Kumaretal.,2001;Guignabertetal.,
2005).BALisatechniquethatcanbeextremelyusefulinthebio-
inTP,IL-1(cid:3)andIL-13(Fig.2A–C).Troloxdidreducethedepletion
chemicalassessmentofpulmonaryhealth(Henderson,1989).This
by SM of total glutathione levels in BALF; however, due to high
techniquesamplestheepithelialliningofthelungs,whichisthe
variabilityofthisdataitwasnotstatisticallysignificant(datanot
bodyfluidclosesttothesiteofinjurycausedbyinhaledtoxicants,
shown).
and allows for the evaluation/quantification of the level of cyto-
toxicityorinflammationinducedbyatoxicant(Henderson,2005).
ResultsfromtheanalysisofBALFwerelessconsistentthanthePF 3.3. Histopathology
data.SMcausedasignificantincreaseinTP,IL-1(cid:3)andIL-13.Apro-
tinintreatmentpreventedtheincreasesinTPandIL-1(cid:3)production, Histology data show minimal alveolar effects caused by SM,
ilomastatpreventedtheincreasedproductionofIL-13,andtrolox which is consistent with previous reports using this model
treatment did not significantly prevent the SM-related increases (Anderson et al., 1996), while damage to bronchiolar regions is
Fig.3. GrossviewofinjurytoratlungsexposedtoSMorEtOHandremoved24hpost-exposure.(A)SM-exposed(H–heart).Notethegrosshemorrhage(arrows)onthe
leftlung(justabovetheheart)andontherightmedialandcaudallobes(lowerleftoftheheart).(B)SM+aprotinin(H–heart).Notetheabsenceofhemorrhageinthelung
lobes(shownarerightmedial,caudal,accessoryandleft).ThesuturepicturedwasusedtosecurethePE50tubingforlavage.(C)Control–EtOH-exposed.Asmallsectionof
theheartisvisibleontheextremeright.Nohemorrhageisvisibleontheleftlungorthethreevisiblerightlobes.
D.R.Andersonetal./Toxicology263(2009)41–46 45
thesignificantlylowerbronchiolareffects(Fig.4),whichindicate
less direct injury to airways, and in clearer airways, which was
corroboratedbythePFdata.
Ilomastat is a broad spectrum matrix metalloprotease (MMP)
inhibitorthatexertsitsinhibitoryeffectsonMMPsthatarechar-
acterized by a zinc binding motif in their active site. MMPs are
involved in the degradation of the extracellular matrix compo-
nents,growthfactorsandreceptorsthatareessentialforhealing
(Schultz et al., 1992). MMP-mediated degradation of the extra-
cellular components results in the activation of chemokines as
well as MMP-9, which together produce inflammation and con-
tributetothepathogenesisofSMtoxicity(Sabourinetal.,2002).
Fig.4. PercentincidenceofhistopathologyinratlungsexposedtoEtOHorSM24h
Guignabertetal.(2005)foundthatMMP-9levelsincreasedinBALF
post-exposure.Incidenceofpathologywasnotedforthefourrightlungsections
of SM-exposed guinea pigs, which could be associated with air-
andthenrecordedassectionsshowingpathologyversustotalnumberofsections.
N=6–8rats/treatmentgroup.(*)SignificantlydifferentfromEtOH.(+)Significantly wayepithelialdetachment.Severalstudieshavedemonstratedthe
differentfromSM.Alv–fourmeasuresofinjuryinthealveolarregionofthefour abilityoftissueMMPsandilomastattoinhibitSM-inducedMMP-9
rightlunglobes–alveolarepithelialnecrosis,alveolarexudate,alveolarhemorrhage, activity,inflammationlevels,andairwayepitheliallesionsinguinea
alveolarPMNinfiltrates;BALT–bronchialassociatedlymphoidtissue;Bron–three
pigspretreatedintratracheallyorsubcutaneouslywithdoxycycline
measuresofinjuryinthebronchiolarregionofthefourrightlunglobes.Themea-
sureswerebronchiolarepithelialnecrosis,bronchiolarexudate,bronchiolarPMN (Guignabert et al., 2005), microvesicle formation in human skin
infiltrates;Edema–perivascularedemavaluesinthefourrightlunglobes. cells treated with ilomastat (Schultz et al., 1992), and reduced
eye opacity and bullae scores in rabbits treated with ilomastat
(Schultzetal.,1992).Wepreviouslydemonstratedincreasesinpro-
much more severe due to the highly reactive nature of SM and
inflammatorycytokinesandlipidperoxidationinratsfollowingSM
itseffectonepithelialtissuesoftheconductingairways.Aprotinin
inhalation(Andersonetal.,2006).Fig.1showsseverallungfunction
andilomastatbothalleviatedthePFperturbations;however,only
parameters where ilomastat treatment clearly ameliorated SM-
aprotininreducedtheobservedpathology,bothgrossly(Fig.3)and
inducedchanges.Pembertonetal.(2005)showedthataerosolized
histologically(Fig.4).Troloxwasineffectiveagainstdevelopment
ilomastatreducedMMP-mediatedchroniclungdegenerationand
ofSM-inducedlungpathology.
thepermanentlossofpulmonaryfunctionthatisassociatedwith
emphysema in mice through the inhibition of macrophage and
4. Discussion neutrophilrecruitmentintothelungs.Inourbiochemicalanalysis
of lavage fluid, ilomastat significantly reduced levels of the pro-
Inthecurrentstudywehaveevaluatedthreecompounds,apro- inflammatorycytokineIL-13comparedtoSMalone(Fig.2C).IL-13
tinin, ilomastat and trolox, for efficacy against SM-induced lung isamediatoroftissuefibrosisandislinkedtoMMP-9levels(Leeet
injury.Thesecompoundswereevaluatedwithvaryingdegreesof al.,2001),whichareinhibitedbyilomastat.WhileFig.1showseffi-
effectivenessbyWBP,analysisofBALFandhistology.Ourresults cacyincrucialPFparameters,thiswasnotsupportedbyprevention
indicate that aprotinin was the most effective of the three com- oflungpathology(Fig.4).
poundsfollowedbyilomastat,andtroloxwastheleasteffective. Trolox is a water soluble vitamin E analog that is capable of
Aprotininisaserineproteaseinhibitorthatfunctionsbyirre- penetrating cellular membranes where it can protect the cells
versibly binding to proteases that contain a serine amino acid from oxidative damage. In the treatment of oxidative stress, the
residueintheircatalyticsiteinhibitingtheactionsoftrypsin,plas- best antioxidant depends on the specific molecules causing the
min,kallikrien,elastase,urokinase,andthrombininanincreasing stress and the cellular or extracellular location of these specific
dose-dependentmanner,respectively(PutnamandRoyston,2003). molecules (Naghii, 2002). Since lipid peroxidation is considered
Theseproteasesplayimportantcellularandhumoralrolesinhemo- a component of SM toxicity, an antioxidant that is active in cel-
static dysfunction and activation of the inflammatory response lularmembranesmighteliminateoratleastameliorateoxidative
byactivatingcytokines,coagulationfactors,fibrinolytic,andcom- membranedamage.Bhattacharyaetal.(2006)showedthatinSM-
pliment cascades. Prior studies using our model have shown a exposedhumanperipheralbloodlymphocytespretreatmentwith
time-dependent up-regulation of gene transcripts that are acti- troloxsignificantlyreducedcelldeath,lactatedehydrogenase(LDH)
vatedbyserineproteases,suchascoagulationfactorIIIandtissue leakage,andperoxidegeneration.Trolox,ip,hasalsobeenreported
factor (Anderson et al., 2000). Aprotinin (Trasylol®) is an FDA toincreasesurvivaltimeandprotectthelungfromoxidativedam-
approveddrugusedintransplantandorthopedicsurgerytoreduce age following SM challenge in mice (Kumar et al., 2001). Using
perioperativebleedingandhemorrhaging.Inrecentstudiesusing ourmodelsystemwehaveobservedareductioninBALFLDHand
thismodel,andasdescribedhere,wehaveobservedthataprotinin (cid:4)-glutamyl transferase (GGT) levels and a reduction in peroxide
was able to control reactive oxygen species generation (Holmes generation following SM inhalation with concurrent administra-
etal.,2006),eliminateSM-inducedgrosspulmonaryhemorrhag- tionoftrolox(AndersonandHolmes,2005;Schrothetal.,2006).
ing up to 24h post-exposure (Fig. 3), and restore lung function TheuseofantioxidantstoprotectagainstSMlunginjuryhasbeen
parameterstonearcontrollevels(Fig.1).Theroleofaprotininin investigatedbyseveralotherswithvariedsuccess.McClintocket
maintainingalveolar/capillarybarrierintegritywasalsoevidentin al.(2006)usedintratracheallyinstilledliposomeencapsulatedN-
reduced TP levels (Fig. 2A) in BALF analysis. Further evidence of acetylcysteinetoreduce2-chloroethyl-ethylsulfide(CEES)injury
thiswasshownhistologically(Fig.4)wheresignificantlyreduced toratlungs.Wepreviously(Andersonetal.,2000)usedN-acetyl
perivascularedemawasobserved.Thisalsocouldbedue,inpart,to cysteinewithsomesuccessagainstsulfurmustard-inducedlung
thereducedBALTnecrosis.SinceoneroleofBALTistodrainexcess injury in rats. Wilde and Upshall (1994) evaluated several cys-
fluids,plasmaproteinandparticulatematterfrompulmonarytis- teine esters that provided some protection against SM in an in
sue(WitschiandNettasheim,1982),injurytoBALTcouldleadto vitromodel.Inthisstudy,troloxwasgenerallyineffectiveagainst
edemaandrespiratoryinsufficiency(Andersonetal.,1996;Baum, SM-inducedchanges.WedidobserveareductioninSM-induced
1974).Histologically,protectionbyaprotininwasmostevidentin changesinthelungfunctionparameterstidalvolume(Fig.1A),end
46 D.R.Andersonetal./Toxicology263(2009)41–46
expiratorypause(Fig.1D)andenhancedpause(Fig.1E)withtrolox, Calvet,J.H.,Jarreau,P.H.,Levame,M.,D’Ortho,M.P.,Lorino,H.,Harf,A.,Macquin-
buttheseweretheonlypositiveresultsnoted.Thenegativeresults Mavier, I., 1994. Acute and chronic respiratory effects of sulfur mustard
withtroloxinthisstudymaybetheresultofthesinglerelatively intoxicationinguineapig.J.Appl.Physiol.76,681–688.
Casillas,P.R.,Kam,J.C.,Powers,C.-M.,2000.Serineandcysteineproteasesinsul-
lowdoseofdruggiven.Wehopetoaddressthisdeficiencyinfuture furmustard-exposedhairlessmouseskin:Enzymaticactivityandinhibition
work. profiles.J.Toxicol.Cutan.Ocul.Toxicol.19,137–151.
Thedosesandroutesofdrugadministrationusedinthisstudy Cowan,F.M.,Anderson,D.R.,Broomfield,C.A.,Byers,S.L.,Smith,W.J.,1997.Biochemi-
calalterationsinratlunglavagefluidfollowingacutesulfurmustardinhalation:
werechosenbasedonwhatwasfoundintheliterature(aprotinin
II.Increaseinproteolyticactivity.Inhal.Toxicol.9,53–61.
andtrolox)ormanufacturersrecommendations(ilomastat).While Cowan,F.M.,Broomfield,C.A.,Smith,W.J.,1991.Effectofsulfurexposureonpro-
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Cowan,F.M.,Broomfield,C.A.,Smith,W.J.,2002.Suppressionofsulfurmustard-
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Emad,A.,Rezaian,G.R.,1997.Thediversityoftheeffectsofsulfurmustardgasinhala-
willevaluatethemusingthesamerouteasaprotinin.Wealsofeel tiononrespiratorysystem10yearsafterasingle,heavyexposure:analysisof
that aiming the treatment directly at the site of injury, i.e., via 197cases.Chest112,734–738.
Guignabert,C.,Taysee,L.,Calvet,J.-H.,Planus,E.,Delamanche,S.,Galiacy,S.,d’Ortho,
inhalation,maysignificantlyimprovelungfunctionoverthesys-
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