DTIC ADA423257: New Approaches Towards the Elucidation of Epidermal-Dermal Separation in Sulfur Mustard-Exposed Human Skin and Directions for Therapy

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by  Defense Technical Information Center

Farts Faprorer

REPORT DOGUMENTATION PAGE OMG No, 074-0188.

atv tne! Banaery 2004 Benual (17 Jee 2002 ~ 26 Tee 2008)

Mow approaches Toveras tee Elacidation of epidersa-nermal | Eawpr?-02-1~0206
Sepcration in GuLfor Mentard-Exgoaed Kumen Gkia and
Directions for Thecpy :

Eie as 20040602 034

| 2280 aa Ridewizk, the Netherlands

| etme nebo be0 0
[= Sansome rmomroRne FE SPORE | ORTORITE
i

‘ASEICY HAMEISI AND ADDRESS(ES)
[0.8. Army Meies) Resoarch gma watertel coniend
Fort Detrick, Maryland 21702-50:2

7, SOPRA ROPES
Origins) conzaina color plates

ALL DIC reproductions will be in black and vite

| aygeawad Zor Pubite Release; Dlatributior. Unlinited

TS ARRAY Ta 2D WT
[teraventeacion in hunen akin, due ta ex vive expamsre to MD, can be tacked ty 11
‘ydroxamate-based ink:biters of Wire and relaved enzymes, Z| furin inhlsizer ad 3)
| peavesgase inuibiLor. The observation Unstone of he bytevxnmite-based intibitors, 254,
[Gan he applied unti- -th after exposure to AD suggests tha: non-ugent core of 39
cetvelties in pevslbte
Daring pathoganea!s of ND-induced microvesicntion the expression ef lacinir-5, ore ef the
key protelne in eptderma? dermal asrackvent, 16 reduced, However, a'thoug lantnis-§ ig
tecget proteda for Wipe wad related eazjoes, these eneyast grr not rorposible for the
Georense 1 lominin-5 gaprersion. Horeover, epiderasl derma] attochment in still posalale,
in spite of rodceos anounts of laminin-S. Cleavage of otver attacknent prateine vhaa
Iuntniaes by Iipe and relacee etaymee Pidbably causes the hcesk elweeo epidermis. end
deme, -xpedinent of tke apoptotic cell death process appears to be alez very efters
in uaiatalnieg epidermal-Carmal adseelon. The oheervation thet ceapere inhibitors prev
Eeagrentatior of keratine 10, 1€, acd 17 in wD-exposed MEX may auggest That an intact
| Jeytookototon of keratin filmiocts le gecentiel tor eptdarmal-dormel schorion, The
Observation that caspase inkibiters provent fraencncation of Kerctins 14, 16, and 1? 2a
aD-expased HEX may euggest that ar intact cytosxelezen of keratin /Llamants 1g exvortiel

fer opidesye] dermal srtochmont
futtur msterd, vesication, baraa akic ex vive. matrix- 23

netacloprebeinice inkiniter, eretcanias, apsptazie, human apidezcel
Kerarinocytes. keratir, fragusntetic

TF. BECURITY GLASSHIGATOW | To, SECURTY CLASSRETTON | 78 SECO” CUABSINGAVI —| 20, LITA TON OF ABSTRACT
orneram ‘oF mie race ‘oraastascr
Gnelacaifies Ucelzasitiog Telassitice i

ised

EH TEDDIES ara a av 257
AD

awasd Nusber: DAMDL?-02-1-0206

‘LTLE: Now Approaches Towards the Elucidation of Epidemnal-
Dermal Separation in sulfa Mustazd-Rxposed Hunan Ski
and Dizections for Therapy

PRINCIPAL IWESYTGATOR: Marijke A. Mol, Ph.D.

CONTRACTING ORGANIZATION

TNC Fring Maurite iabozatory
2280 AR Rijewijk, The Netherlanés

REPORT DASE:

January 2004

WEE OF REPORT: Annu

PREPARED FOR: U.S, axmy vedical Research and Materiel Conmaad.
Fort Detrick, Maryland 21702-5012

DISTAIBJTION STATEMENT: Approved for Public Release;
Distribution Unlimicea

he views, epiniose aad/er ¢indinge contained in this report are
those of the author(s) and should not be conscrued as an official
Department cf the Army position, policy oz decieior unlues so
designated by other decumentation.
‘Table of Contents
Cover

si

“Fable of contents

List of ahbrevistions

1 Tenondustion

2, speci cto
3.Realts

4 Discussion

5. ry Renan Aes plishnents
6 Reportable Outcomes

Cassone

8. References
List of ahbroviations

ADAM.
AEC

ont
chats
20-PAGR
DSi

pri

HD

erp

nse

HER

a

re

Da

Kea
KRM
MALDITOF
uM

mit

wor

us

so

prs

pt

Rowe
RNase
SDS-EAGE
race

Tris

A isinegrin und et
Bamion-S-xjoaiban
Visemtnt meabeaie
-CholanidaprplialiahyLananoriol J mopane elfen
‘Tsnediensiona lyseremide cel slectrpsessie
Trespibesmstene

1a Dihiaueis)

Sutter mustare

erste diheresidans

Tea Sige poten

‘roan opidaral KerwSnonsts

Teoelestie four ng

Lnobilivg pT piadione

‘ilodilen

Kerainooyts roth medium

eraneyts esa edison

‘Maia assist aes descapionton ation =
Micro

atimolar

Spree

fight

Mis ett
Mass spacey
‘Molecule wsil
Phogpkate hued a
Ihoeletie put
‘Redvelom ceralibledetrgent saga
Ritomzlewe

Baling dx tf pyran gel electnghovess
nove cecesis ftslgter couse ey
“Triihy rong pameneme-nane
1 Introduction

‘Tho sudios desert inthis sent are past ofa esouc pvt (award umber
DAMD17-02-.0246; aie lo obtain alagurte meal defease agus inuy eons) by
>sparure tala estar (HD). i projer il clucidat> the mezhasism behind =pidermal
‘Ser sepia ia IB-esjoved tin kia ero lin a asa ergy again Mitr
fecmason. Loss of ts ras"macntcompetsace othe basemen: mr: (ER) proteins
postulated ta hea specific cans of HD-indacodvesisaton of the akin ad dietnboe. bance
Trtoven odnaton and degredation of hots pesos has hocaaopgested contin
Thee, anleru prove reruvtieaion, egiadatinn nM grote dhnold he dished
saa ke pration vould Ee ease

113s known at BM proteins are specially degraded by mix metalloprtenases
{MIT a Femi of enzymes at isle in narmalrortaer athe catrselol: rat
‘lene nd Steller Stevensen, 1999: Nugise aa Woessner, 1999)-Ta te ese studs,
‘ivione ints of MMPs tata on repel ty race AD-induced
alcoves ast by preseratton C8 roe. Cn (a llr Ral, umsnece oF BM
roins eu Iso bs obisved by sasuring hee symtess -inlucedepaerl cell at
‘ei eauis ahold up of protin sytbsce Basa en indiccions Cat apoptosis is nvolvd ia
TMieduced ost iejns Resin ta, 2% en serra eed wehethoe
Week ef agnosis hy enapase inbiiinn won Revs hentia ese vhs raitenance of
Te 2IM.

‘Lainie ew kay protein in epdermal real atachacct. dit hs been éseize
thar the protein fe ubte for MMP and slat eroyrien (ane ot a. 199% Pie et
2003; Veic et, 2008, Te vse canes inthe express velar § Flowing FD
‘exporeo end he porsbleetfecte ef nition af MMPs and carpus, ranurobstecberial
rs al the prs patra af contol and TID-treuted ecliured

(CHK) gle ayyoocet in revel ela cea
‘veuring ler MD exposure. Inthe rota profile of LD-expose LICK, esi Urgusesuf
kevacne 14, 16 und 17 ware ahrere, Sine ragmention a cytoskeletal proens ert
‘apne, ae teste ehetheefmation ef tess Epmenes coal be peesend wth
LW:0r of apoplosh.

human spiral Rerinne

2 Experimental Metheds

21 HD synthesis

HD har
east 07%,

5 thesized at TNO>-Pring Maus Lahrarvy and hes a pry out

22 Exposure of sin pleces to HD vapar and human sox organ euttre
uronn manemony shins oblaied fom easmetc surgecy wit informed wave of
‘he pie
iunon mancmery skin has boon oxpasod to satureca HD vaper at 25 °Casing a
vopac eup dovieo sx dsseribed cals: Ql ct, 1988. Ths duration of exposuo ee BD
opoc wae fle minut, umes tare ndiatod. Oepsa cuore of naman skin ws
sesnlaied 5 decid ly Wat etal. USS. Sk oes af 25 a were Acad with he
onal sie denn in Kertinceyte hal wediam ¢ BiaWhinake: Baap, Verses
‘supplemented wi C¥Cl oa ial vonceatabi of 4am (EDMCas Il rail of
(seo loser plas) and icubatee ot 37°C in an ctnagphere of Xin ay. Pos
-exgoeeminenfation we fe 42h, ules tara dite

‘The MM oboe DS a fu! Dis bioock, Oxemd, UZ the ponsapase
inhibin 2-VA- FAAK (Signa Aldrich Chom Zwijncrosh, The Netherlands), she MME
LAMP 9 ato (Caio, Ss TDiggs, TA, Mion oF MIP 3,
‘esibteeT, Calhioehens, Sm Diegu USA) ad intbitor uC MME, 2
11, Cabiochem. Sen Diego, US} were dizoled in DMSO and date i gn clare
‘odium nthe deed final concentration? The final eanean an nf DMSO was aby 1%
TlaeSwin hi ie RVER CHK (Baste ACY, Rural Silat 3 it est
Besolved in ecgan elure mening he dese Bl concen

23 Hisologe

awavisbin pecs wee se gvorigh at 4°C 4 24: parson
hoophnte buffed swe PBS) They were sine iu 70% chaucl ull euleleg i
unin. Sess wre sd wil earteaghtrinwtesaenned by Ligh eros
Pruwugciphs were taken 3 4 dil crema (Olympu C5080 Zoom.

24 Fosmnnistachenetry

roman sti psees woe feecen in Tissue Tek OCT exubeMiny compevea at Sp
ceryetatsetions were coe prevent deren of Laminin iéas with eryceoctions ere
‘nencn named afar ening ar kp ficzsu-int tte acess ng. Mel Naeking wih
nea-apife sets, cations so inubete oxen aC wil masons ee ae
haat tie (claus GBS, followed by 1A baobnon wt ream tempercure ea
biovuplced goat aat-merse 136, The irmauneperoxdate ezine was pevfomed using a
sin horseradish poroxidass (HRP) conjugets fxd Sa nine--cthoarhana (ARC Slides
‘wero conxsmtined wth Raorecexylin and maonteé un see ernst
sero performed by omitting ts primary acho doing the Fist ication, D924
seers taken by a gil sara (Olyzrpus C5050 Zoom)

sever.

23° Rinanam epidermal kevatiocyie (MEK) enttare and exponie of HEB 1a HD

Fis pssege HER oan en sf ued Fs heise ain
(EGR Eee Farag, Vern, egw Lu sy ce celal reponse
Yo TID, he els were exposed fr 30 sat, 1W oe 750 LE

One dy ater reaching cufluencs, HEK sero expesed tH (2.5 reall ofa Gast
Slater plat) min at 23°C Stk anda HE ve fea’ vain hy anes
2nd Giuled innusditely Uefe eve i KEM obi De dssred workig concen, Te
find voncentrados of acetone in the ocabition ced wus 1,

Folloving 2.4.8, 15, 2¢, 32 and 42 of eac-enporate incubation with fssh KO

els

‘He enlnes we rene frie U mesons poljacylunide gee up oresis PAL"
Tues dove well wb decaf estmeat were poole

Te wey whether pocinfugrsots were do cleavage by oacpases. HEK cultuee
‘were incubsted th KGM conning 20 UX ofthe cenpaie- and inh Z-DRVT
{POLK or 20M of the cuspaze- iehibitorZ-VEIDSMK, or 20 pM ofthe pataspase
fate Z-VAD-IK (al cumpie inkisitees wee fon Sina AlAsh Chains etna,
‘ie Netherlands. Tie espone nbs see added 3h vio xe TT exp and dng
the 24h parcespoare pnd.
26 2D-PSGE
(Coll sveve solebilizad in U'mokd Tris bottx ‘pH 0] comtinin 1.5% SS, and the
lysate wa boiled ineodl at foe [Gmina 98°C fie coning cw er he Ieee wore
inerlatea for 2h at 4 with 100 aM DTT, presse ichibitr cosh (Complete © Mini,
Rocky Diugnuis, Manchine Germany and DNase RNase (Roche Dispaestcs,
‘Murntio, Carat). Aer sonication hy fone muri of 5, pena Woe stated a
1 vchlaaaoetic aid Sn ioe el nto pert ith 25 8 DLL.
‘ezsbes rice wilt ie-cul auton und subsoyuenlydisclvedin95 BE orca, 4% CHAPS,
115 1PG puter ot 25 mv [YT Prem content re dae with fhe RAN psoeha
any (Diva), Sanples of 150 yg poten ore les om 13 om trorabine IEG tps
{Amershars Rinssenes Bjsteah, Roose ude, th Neila) oles feng 8
ie ut a 20° Cy 33,8KVb on ao IPGphceappartus afore SDS-PAGE, the feared
BG strips wore equated, raced and alylaad'n 3 hater contin’ np Moe 50M
“hicHCL pH88, 2% ST, 0% ahyoml il ier ID eg DIT or 25 mig
Ioagcels nid The hens ee eae ese iene om
Tanigrmons poly ndde gle 10%). "Uhe SOS-PAGE wus run ua eoustmt oo
2 .a4gel a 20°C, Gels were sti by amas spoctomry cmpaibie ster ezrin, ¥en
tal, 200. Compursive analysis of sein pats has son serforer paral hy <3 900
arily by using ImcgeMaer 27 Fie env (AnerslansPcanca Ditech,
Rosendaal The Meters), Tue each compris ut scat faur pow gel of ach samgl>
‘were ued, Foredaazes in pecs spots obs consiéaod a genuine difesnsze'n potsin
eapresion we apache falling eriterine they shold show sindlae grate changes 2
capreaion inal exarinod acl rigieving fo: ue staple,

27 Protein identification
Sines the pereantapo of suscostily emi ogg in go label wesume
ose Toor excited gel epee sere ge biuCa: (edetber, Germans) irae
sgecroneteeeuitcatn, However tir id of ited proteins ex nt ete hen
buts. "Theroiess, we hewe opined eur pesinusl sed praedive fox pete idetication,

specifically ith egard che wypsin some ad the rutin ype,
Spot nf ives were exis and eut ue 1-2 m3 pieces. The paris wee
veoshed and eehrdruted belo they wore digested widh ICD ng type ageing pole

(revnegs, Leder, 'te Netherunés) i 25 mM NACA overnight a1 47°C kes
(Gensen ot al. 199. The peptidar wows canal wie will fon wid sa wetacte
{21} end ths extate we pooled al ded ina wacuuanvestellue, ene desuking the
iotides weve dinsoeed in 39% fureae wend, Desting was permis with Zip
anders uf CIS (Milian, Arete Zit, The Rather) Pie mss
Fingeupink were date yal ated er suri uation (ALD tia
of Might (TOF) MS on Bea er intent using -eyane-thydrexyeinnaric eid a he
snairaThesezuling spocta geet of mates that st kad ino te Profound NCBD
program. Ssarch eta were 0.1 Da ats aacuracy sr. wih of at eos far ype
Busine’. The ProPoutd ene: “7-vai, nding tbe quay ofthe dative eet ecuks
fad tobe Fighst San 0.8. The SWISS PRO du abate ‘utp eatenpasy ches ou) war
se aol ineerasien en p, MW un emino ac cempasiin ofthe ier ed

reine
3 Results

4d Effectiveness of potential therapeutic agents to prevent microvesicaton

AEE aos
In prsvioes exporiment, te efzctivs concentration of B94 n proverting
vsomesa la Bea aa588¢ Feu a Lyf 0 igs. Ta akin, is aon

bat RB94 canbe se 2 eae 8 hale: enpce sr tw ID ¢Mel, 2003), Ia be peel <i,
ita vestigated whether 27 longer Lag times were alleed betwen LD expssure ad the
agen af BB 94 tn the oracle muzium. wea lansing ths et fess he
Iehiitou.Thor‘ore, BAS was alded tue organ cule noc in # Fn esncentali. of
2D gol dp Ma D,& 14, 1B aed 24 hater the ia af Ihe pos expoewre cuba
[Mire its ehown tac 20 yettl BUDS canbe ds a ae os 18 alter expec ee UD,
‘eth eoring ie iibting ec: an epi

LZ Bute ttledor

Feria eomvort:pra-MAIP2 and poo“ MTL-MOP inte dcr activ forms, MMP-2 ar
1MT:-MaMP rxpoctivey.Aplisiton fa Fain inhibitor nigh Mersfosrednes pret
epidernil derail vrurati of ski {somos ID exposure. The (urn inbitter Doe RVR.
(CK ts been adel! Lo cle zusium credits be uno Se put eapuess
uta, ia vooentznivas ol 25, 50, 10), 250 or SUM pi. ete -dopentent dation ot
smrovesioutinr i obsersné “nthe prssonse of 50M -rin nbibior the epidsrag ane
deri ofthe skin were ot tom pen (Figure 2,

urn uit ise 1 SOOM 8 Wwe evan Sh tee,

BLS Ini of MMP-t, 3, - and flavor 15

A yatznt oad rag ‘ohiar of MIME I, 9, F and 9 (N-AyCIONy |
meszengbsnzenseatfanngT-5.£-dimethyl |S]. pigeavine-2-arbosanaieyae wht hy
Destexpocuie eg cute madiun in eocertarin rng fea Oa » 200 wil Tis
[MIP inhbitos igcoved epider dered actual i sig a“ er LID erpesare but fll
roleuion agus: Hinducod microtsiowon sve at cahiovd ae Highse:comcetstion
:ec2 (Figure 58.305. Th ohio as ws ba cove“ human Ski (it shaven.

Fndet Sabian of MAME, 2-2, 2-13 Sobibor 1p

A homophe-nydroxemic acid based besud range nuitor of MMP -1,-3,-3.-Fand
13 was add to th post-expeeuo organ clare median ineoacoutrations rangi fron 3. —
11k). A dose-dependent seduction of microvescatin was cheered ar tl potstinn
sain EIbitdncod micensieann wt otcinndl with concevetione af Sof a pha
(Figure 80, 3), Tas align ise ws wet tone To bu sin esa

ALS MUP2NREPD inhibitor

A gotenc inhibits ef MMP a 2 as MOMP-9 mag aed 1 Ga po espns
vg calle atin iene atins ging sun 12-82 afd This alle
apps? co be ond ete bora kia ul envenkraons of 25 yg! and igace (nt shown
‘Altbe pon-xde concentration ef 12.5 yeh the inhibitor as iefetve wid regard to
recoction ef micrevessctin (Figie 30),
S.A Panaypase inkihian
‘exec ee coll deuh of keinocy'es he gin eullre sca Pes seppeene
‘rian fel cages enayies, ZL ALLLIMK. be inhibit wis ued immiaately
at he sia the prsr-ezpesosincuberinn, a cenceneions of LA 25, St 100.9208 eM
belly blockee maeesuatluais user wtb 1M 2-2 aul ghee Ly aun,
‘bin adestan of mcortc sells fs obea:vod Usha
encase ibe Wace 206 pA was ote roth harran sk oe

stent

5 in HDeindduced microvesiction

22 Role of lamin
“Toiasesgste wea changes i it Sears ae glace dig he
Jevslopmancofierasesicss, naman skin pice hat were cxsosed for 3 rin te MD vapor
ser excmind hinologica’y aed imannaisachericaly 14,8, 16,24 4 4B of pst
ose inc shaton Hisogially ie: siges of epidermal cel death re seen at 16 baer
naar, whan eresiarin is eve at- I postexpoone Figure 5, 3, $F, 3G
‘Tae imauaosheice’ ani, palen foe lana 5 sins tal anni i abel presat
te athick fe alone Ue epee dere janetina in vutol skin (gare 32, ALS
Follevirg eecaurs to HD tho ening incenity o aritin-5appcar te ho ed wed and.
1th Tet as vs hin ass abs sd Pape Hewaser, hon
_ncroveseuea ts bled by BUDA, fr itor ux pone. fab, tbe expres
lamciin- i oe inyaree esmpared sp aneated HD-expmod skin igure fi The
ics th ar oni he fie session that ee nee Iara
tre tei a bistulugia? sete of UD -exporel! skin det ee i
Inbibilors ne mieroesioiunis cberved gw / 2.4.

eas

13 Differential deptay of PEK protebus associated with exposure io HD.
Ceamrarse quaitaiveansesisf gos nina at 8, 24, 32 and 42 Raf nenhation
foowing wastes he I pM HT) onsale sais
exqursi el were nab ihe ste MeL Asien saving aa sailing
method, only proteins spo tu: consstnly show a secknsat-Sopeadsnt changin
Imensiry ceampared ta cnn os nfzed a8 HD xpos eat. No diferonecs woes
1 palenu ubteined fra IEK expuse c 10D urtu 180 MTD, uber
were clilfences cberved fo prema of elle that ere leet at 24,12 or 42 post-expone
sesuts not show. Yoeratre the silver sane: ysosin expression purrs af HEK expeeed
“2 106 uM HD ofszined at 24h post-expesus is aksn ax ypical- Tn tis proton safle
several pos ate ehserved fat as related to MT expoas. Mos of Bem 473 Seca hs a
ilb pLAS~ 5.5 an MW 20 30D, Proein paters of contol and UD-reaed TICE af 24
‘a poe-exposne ‘hata found to be typzat for this arcu ao swt in Higa 7.The pxotin
pots ha ae india’ wth arms are qualtrve renter alata. Th
{ie pts es beets oubjtel eie t ngenperaneuli i he
‘eaten uf twelve uf bem, Table 1. xcept Loe vo, he tound pets were
dogeadeion produce of th eytcaksht!kerarne 1,1 and 17 thet ae press én
sndiffroncatedculusa HER. Spats by 9, h ad tar fesgnr is of Ie Thesis 1 14,
Peptide mas “ingeay'nng of apes ad yell ypieageutas oa ated wk
si auld caqueeces i the Cee! porto the rosa, heres the typi Hagens ha
were beereliz spol gd and i ooassed ofemine acid wees tic oceur exclusiva
fie 8 terse part of KI4 Pep ile ms izgeprinting of spots ay € and treveuled ta ey
re Cas! Nermanol ngaxns of ype {eran IT (173, Spots unk wee identied
(Cad 3-teemina tages cl ype Leal 18K). ily, spe a em ba oe,
iesntlied us beat shook protta /HSP) 27

‘The prtoee inthe ers msdiaa of the spose nts Z-FADEMI, 2:
DEVO-FMG, ur Z-VEID-FMK dugg pstenpensee ucubaiua prevent he aypecaase ul
the keratin ugcnts. The HSP2 spss fandmrorzincxszzed in Ca possnce cuss
inhibits. indica tha: hee appeaancs is independsne of cespaie activity Geum 4),