ARRI (RMED FORCES-RADIOBO VSR UREPORTS APRIL-JUNE i989(U) ARMED FORCES RADIOBIOLOGY

UNCLSSIIED RESEARCH INST BETHESDA MD JUL 89 FG67 M

UNLI F/G6

~3 6

111111140

liii - II"- -

AFh RI REPORTS

00_ _ _

(N

April

May

June

1989

C LE-CTE-SEP 25 1989

Defense Nuclear AgencyArmed Forces Radiobiologv Research Institute

gesd9 arylpe 0 10 rj jApprvedU fo p ibi r if;s &srbwo t imfI

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SCCUMITY CLAUIPICATON OF TWIS PIAGE

CONTENTS

Scientific Reports

SR89-15: Bogo, V., Zeman, G. H., and~ Dooley, MI. Radiation qjuality andrat motor pe(rformnce.-

SR89-16: Gunter-Smnith, P. J. -Gamma radiation affects active electrolytetransp~ort by rabbit ilcuin. IL. Correla tion of alaniie and theophylline responsewith nmorp)lo-~v.

S 1? 89-1 T: hunt. NV. A., Rabin. B. M.. Joseph. ..A., Dalton, T. K.. Mzirray.WV. E ., Jr., andI Stevens. S. A. Effects of iron particles onl behavior and~ brailfunction: Initial studies.

SR80-18: Joseph, J. A., Hunt. NNV. A., Rabin. B. M., and Dalton, T. K.Correlative motor behavioral and striatal dopaniinergic altcrations ind lce(I b.N"Fe radiation.

SR89-19: Kalinich, J. F., Catravas, G. N., and Snyder, S. L. -- The effect of7 radiation on DNA inethylation.

SR89-20: 'Mickley, G. A., and Fergusoui, J. L.7 Enhanced acoustic startleresponding in rats with radiation-induced hippocamlpal granule cell hypoplasia-:

SR89-21: Pellinar, T. C., and Neel, K. L. Oxidative dainage in the guineapig hippocainpal slice.

SR89-22: Radhia, E., Vaishnav, Y. N., Kunlar, K. S., and Weiss, J. F.Radiation-induced volatile hydrocarbon production in platelets-.

SR89-23: Schuening, F. G., Storb, R., Mleyer, J., and Goehle, S. -Long-termculture of canine bone mnarrow cells.

SR89-24: Walden, T. L., Jr. Radiop,-. ion of mnouse hematopoietic stemcells by leukotriene A and lipoxin 13B

SIR89-25: Walker, R. I., Schmauder-Chock, E. A., Parker, J. L., and Burr. D.S elective association and transport of Carupylobacter jejuni through MI cells ofrabblit Peyer's patches. -ACcession F~or

NTIS GPA,&I

DTIC TAB

Justifica~tion-

ByDistr i lt ion/

Av 11 l'-lity r

Aisl

ARMEDFORCIESRAOIOSIO11LOGVR.ESEARCH INSTITUTE

R{ il1 1 bI\ Rl ,i \R1 I IX.- SCIENTIFIC REPORT

SR89-1 5

Radiation Quality and Rat Motor Performance

\ t I ( )R N3 X I( ) (; It~ AR) . Zi N VNi A\ x\ Nf.R )D I I N'

I' ii l, 1 iri,'it 1 I RRI I ,dauur 1.1 10 liiii Wi'ti.ttra i/

oi icr , - 4-n 'o)

Ii i,. i S SN (IL 1~ 1)i i~ 1i N . NI, Raiation Qulii1\ arnd Rat Motor Pert'0rniart.1 1c

Ril Rc> IIX. .4 1 -3 9s'I)&I

IhC eIleen Ii hI traI IM1iiii1. elCtron10. J..ird netLittllo radiatilits \%ere m,inetiatd oin thetinter 1eril thne ol Ittale pan-ci e rats. Rats t'.cr irradiated Jt aj milineII tissIe diis

't '( * lim) I511 w ii onc ilkth toiloivi IS (i- le\ l rins 40) or I I-Ntl'iiissiA~l~(i \ 5- tint linear iacceator. ''(Ii 2S-Nf\t\,-a phtowin ( -251. it

1111 1FC~IT1at 6-i \t\ issierrt \tieiiitd-iteaii cliti \ 43). Raidiationi etlcteci

a diNki'i inceintor ertoirniancc test II ), slre hased on I -Tit in )0ost\postIir perIltr-uoII)1 - I lie I I) \\C 1 -hl (I to hirlectroits X I (. s or IhremsstriahInL!. X')G (Is r -a photton,,.

andtl ( i% til ntitrii Ili terisl Ot reLaisek bilogWica el1-s it es t podce earls pertor-ma,MC~ st11nt1 I WI111 11-1n tri t1e 1tar 1t rradiatiloll. siiititieant dilrnees eiste hetxe

time lcirit 01is an1d the ii timer threeI Ilds and heIssCI eenC th111msratigad Vileutroit lItids. these'ouiertce tld il lie esplai ed h\ itaeticistc diise distiltiiiit1[01 patternls Ill the irrladlaated

'11111kI he datal iIm pit that ditlereit radia ot uli ties areII tilt10 eqnilik edlleetis t disr-uptingpmet otee sslilt highicir. eIlti ins heHii!i theC lttiist ettI\e and IIltutriin the least. Ill

II Rttt(t ItON

I-ark perl'Orm arice decrement ( .PD) is Ittten produLce.d h\ eXposo rc to rapid, Su per-lethal stni/IngC radiation ( /~ h:lo\%\eer, tt m~as recenly reported that 1-P[ mna\ occuratI doses helIo I te 1 I DX, , (2). When IPTI ) Occurs It uISU alIlI starts I(0-15 mmi a fterrradijation and c.an last anoth lcr 1(0-30 nln. 1 ow\eser. all radiations are not equally

CIIcti \, at prdcn V~PT). I-or Instance. ) photons and bremisstrahlung radiation\\ crc miore ellecti\e ait producing IT') than neutron radiation in pigs and mionkeys03. 4 1.and electron radiation NAas miore ellctive than -i and bremnsstralulng radiationIn rats L(5). While [P1) Occurs in several animial models performing a s ariet\ ot tasksafter exposure to x arious radiation qualities. no comparison exists Ii one animalMotdel perhurm1ing4 file sameC tasK. -1 is paper continues prev iouS wkork (5, 6). and it

describes the elh'cts Of' bl-rImsstralulnz. electron, and neutron radiations on rat

niotir pl-Ornio rice. Ih l bjecti\ es %%ere to establish the miedian etl'ectix dose (ED,,))fo r rat perfornatnce alter exposure- to to(ur diticrent radiation q Ualities and to deter-

ininc their relati e bio lo gical etkect i xeness ( R BV).

\it1i nvlii IituS ."In ti l"i -- vdii

342 tl060i. /EIMAN, ~)D OF

I AB 1:

I IN.\C Iectron a nd Rremsstrahluing Radi at ion Parameters

h(1:/1i Brit %Nit oro1,/imm

Pulse ssidth 4.0 gs 4(0fPsBeam enems% I Icx 18. I 'VVplieati 1:,1curret 0.54 amnp 0.54 amipI )istance 4A4 Tit 28 i11D~ose pulseW 8.3 ei C6% 0 e(; j iPLse~ repetition rie 4.los 60,0)D~ose rate

D~uring pUlse . I) l0(is,nin S.4 l0' (s/minc\ eage 210.)) (5/ml -10).)) Gs ,'rin

Mi[ I HOD1)

N! ; l ale i( rl: CD BR \ X PIlus rats M Rim i o %%i o L\rre used that ss eiihed an) as crage of'4t L 4 istandard error of'the mean ('ii- I. Rats) ( \ Wi this sile represented the age group ol interest

in ,sLr ss ork Rats %%etie maintained in an X \1 XlCX-aeeredcited tlacilit\ i: keeping ss ith the principles statedit) the 01i, ,uu 1P f /1 ( I/C oid, Ic Cf l Idor, '. 111ooio/ prepared] b tlie Instituie of I aborator\ AnimalResources.N Naitional Research Council. Rats ssere q uaranmtied on arrisal and screened for disease. I he\

iicr naiitindin plastic iciilairceson hardssiiod chips changed three timecs sseek and pros idedcoIJnicrcial rodenit choss and acid ss ater I P11 2.5 using coneentrated 1-10) aid /ib'limi Rat-holding rooms,%sere ma11itailned at '() - "I ssith (r' 10', relatise humidits and at least 10 air- changes/h of' 10f';conditioned fresh air. Rats, ssere on at 12 h light/dark f'ull-spectrum e~cle (lights on at 11010).

I a\A Perfrlm ance ssas assessed ss ith the accelerod. a .shock-motis ated test oif' motor eoordinationRats 55%ere trained it) miintaiit position for as long as possible on a graduall\ accelerating. 5-cm-diamecterri d l~ iated I S eII ahoi is grid .hock floor. [ he rotational s loci ts of the roid Inrcreased at I rpm/%. In thetinal sItges of training, a trial lasted until a rat could not maintain rod halance (in s1. Shock sas gis en onl\on trials that lasted less than 3)) s. I raitiniz sessions lasted fromt 5-IS ru in. anil it took an average of 8trainring da\ S to achiCs e stahle pertliin anTce. I hie asci age performance time prior tii irradiation ssas 453s, sN ith performanice as eraged us er three triaIls,/ si sion/suhlect.

/,-\ ,Pr's ,'i/iir ( )nice stahle perlormance ssas aehies ed, subjects ssete conditioned to at shaml irradiationpiiCed sire. Shatm ciond it ion ing lasted 4 rla\ s for 2)) niin 'da\. sim Ulating all aspects of the radiation-dasprofile. Suibjects ssere irradiated Ah ile held in c~ 'itid neal plastic tuhes Q0)) 7 -((,3 cniH. ssith %entilationhiiles in the sides and inc end. I hie open end ssas taped ssien the suh.iect s\%as inside. I-iillos ing irradiation.ear h su fct ss as tested Iniin frioim tile startif e posiiire. More details ahou t the met hids cani he fou ndin Ret. 6).

Ra/iatw it' Rat sLre si ngl i rrad iatedL h\ one olf eiclos i ng: I 8 6-Me\lect cri ns ir I 8.I-. N v p hrcm s-strahlung from a linear accelerator (I. INAC\( I .2-e''o ) -ra photons. iir I .67-MeY ) tissue-kermavkighied mea) L-nerg ) fission neutrions f ,rom a reactor. I issue-kermia sseighted mean neutron energs isthec sseighted as rage of the ntetutroin c-ierg\ spectrum (S). Rats ssere unilateralls espiiser. right side to the

5' C ia ii 10 IitL s I aeii f I ( s; iiiin (Iahlel If the m idl lie tissue rlose rate- ssas ieterm i nedl

INs inseoling an Israrlin fissUe-equisalent itini/ation chuimher ((.5 or0S c Lm)' itt thlL' rnter ofa S-en,-ii i~er istt-LLf isalLn ra pi r tm.ltiiila mher preci sum ss 15 ', () )

I lct ion iiand] hre msstrnh litng N -ra s i rradiat imin ssere dlone using a I I1N X( ss it pa ratmeters listed inI ahlc I. I hie idlim ner'retIeT'IC %%as a laser aligned to the cenitter if each rat. I (ret ri n i rrad iat ions ssere

'10t1 iso 6 rats es p'~ iLITin plaistic: tubhes pl aced iit a ssuii den tahle. I hie .\- ra spL'L t1 inssas piid c'r h\

accelerafi ng thI lefer-ron hea ittua Asatcr-ciioledl cons enter consisting Of four I -mni-thick taintalumn foils.\ lead flattr'ttinur f1iT ()) H 011c thick) ssas alsoi interfaced 84 cin hitt the hean port to imprilse dos,-iinilirmif\ andI to hatrdern the bean. i .L.. to remus r the lo" -cnerg% "taill of the hrr-iuisstrahlUng spectrum.

R,%DI\IION ANI) MOIOR PERFORMAN( 343

Each rat. in its plastic holder. ,,as placed inside a IO-cm-thick paralhn case to provide uniform environ-mental scatter. -\ 2-cm-thick plastic sheet ,sas placed in front of the arras to ensure electronic equilibrium(build-up1.

(amiia-ra. exposures %sere done in a 32.000-Ci '"Co facilit. in which "Co ribbons %%ere inserted in aplanar source tholderl attached to a remotely controlled dlesator. The source "as noirtiais stored in a, aater pool ( 1. • 4.9 -10 fiO) located in a containment room. Gamma-ra\ exposures wsere carried out,,ith the source at a height of 28 cm and centered on the rat. and the\ %%ere terminated b\ lowering thesource into the pool.

NeItron exposures %%ere done k% ith a Iriga Mark F nuclear reactor. with a 15-cm-lead shield interposedbetv een the reactor wsall and the rat to attenuate most of the primar\ -, rass. Rats %kere centered 61 cmfrom the tank ssall and 123 cm abos e the floor. [he tissue-equivalent ionization chambers filled with tissue-equi% alent gas measured total dose. and a magnesium ioni/ation chamber Illed %kith argon gas separatedthe -- ra\ component of the dose. Ihe neutron to -r-ray dose ratio ssas approimatel, 5 at the midline ofthe rat dosimetry phantom. Neutron doses wsere the total neutron and -, -ra., dose at the phantom midline.\in extractor s~ stcin that : -ccd the reactor room %%all ",as used to remos e rats after e\posure. lie system%% as an alunlinun tube that extended outside the exposure room to a position in front of the core and apol\.eth\lcne carriage drisen b\ a motori/ed pulle system. Rats in plastic restrainers \%ere mosed out ofthe exposure room 30 s after exposure.

t'h10-ht,,, mca, 'cm u Measurements suere performed in a 5-cm-diameter cs lindrical acrx i~c ratphantom to estimate the absorbed dose along the lateral (side-to-sidel ,ertical (top-to-bottoni. and longi-tudinal (iosC-to-tail axe, in each of the !our fields 0)). Phantoms 'ere equipped . ith remosable acrylicrods fitted ;%.ith small ca ities in ss hich thermoluminescent dosimeters (ItEDl) or actisation foils sser,placed. 11.1) neasurements %%sere a\seraged oser seseral exposures to determine the dose profiles from ''(o('our test runsl. electrons (eight runs), and bremsstrahlung leight runs). Neutron depth-dose deposition%%'as estimated from the as erage of indium and rhodium fbil actisation ofthree test runs.

it s/io Fhe main performance measure ssas [PD during the initial It(-min postirradiation test period.A modified up'dos in sensitis it\ procedure wsas used in the initial estimate of the 'D._, for each radiationquahtl, starting \.ih an arbitrarx dose. e.g.. 77 G.s ( 1(). If the initial rat shosved FP) i0 min after exposure.the next subject reccised 59 G I f rio [PD occurred in the initial rat. the dose ssas increased ( 1I0 (\ I.Once the appropriate dose range was established. more rats ssere tested at two dose levels above and belossthe estimated E).,. EPD %Nas defined as performance two / scores beloA baseline. i1 so /scores representthe degree of signilicant change in performance postirradiation at the 5'; conlidence lesel, adiusted forindi idual dilikerences. I he mean baseline prescore was based on 15-20 test periods. 5 min in duration.A\cross the radiation qualities doses ranged from 45 to 130 Gy. Probit analysis wvas applied to the doseranges in each field to determine the probits. ED,,'s. and the radiation biological eli;ctieness of eachradiation 1i// . RB[ is the ratioi of the absorbed dose of one radiation qualit\ t) the absorbed dose ofanother radiatimi to produce the sane level of biological effect (1/2).

RFSt11. FS

As stated in the Introduction, this paper is a continuation of previous radiationqtualil. work. The behavioral findings with the brernsstrahlung. electron, and I fieldskscre reported elsewhere (5. 0). The dosimetry work was reported in an AFRRI techni-cal report (9).

I,.. Probit analysis of the fbur radiation fields assessed the efliccts of'each radia-tion qualit', on behaior 10 min after exposure (Fig. I ) (11). The resulting estimatedED,,'s and their respective confidence limits for each original data field are shown in[able II in descending order of effectiveness. The analysis also indicated no signifi-cant difference in probit slopes for the four fields. Thus a pooled field was calculatedand the RBEs comparing the radiation field were estimated. As shown in Fig. 2, pool-ing decreased the slope of the electron probit and increased the slope of the - probit.The findings indicated that electron radiation was significantly more effective thanthe other fields as shown by the following RBEs: 1.6 compared with neutron radia-

344 R060. ZEMAN\ *\Nt [X))tI

S 99

Bremsstrahlurrg I-)U

0 95 ~ElectronI)S 90

2 801

0 7060

S 50- 40

c,, 3C NeutronU 20 4

* Gamma).I 10. 4 5 6 7 8 91012 14

RADIATION DOSE (Gy x 10)

1 1. \ccelerod eark performa~nce de'crement clos-response cur\ cs produced h\ hrenisstrahlung. clec-Iron. -i. nd neutron radiations.

tion. 1.46 com pared w ith ) -rav photons, and 1. 34 compared "with bremisstrahlu ng Xra\ys. Bremsstrahlung radiation was also signiticantl\ more effective than neutrons(RBE -~ 1.21 ). No significant difference existed between the ' and neutron fields.

I £thahilt. No mportalitN dose-response relationship was found in any otthe radia-tion fields. probably because the range of doses in each field was small. The averagetime until death for all radiation qualities was 102 h.

1)c(n/i-dO. M0 Ml~u cr'/ucnif. The depth-dose profiles for the four radiation fieldsin the three-exposure axes are shown in Figs. 3-5. The precision of the depth-dosemeasurements in the "Co0 and LINAC fields was about 4";. Due to positioning prob-lems A ith the extractor carriage and the small size of the activation foils, neutronmeasurement precision was about ± l0";.

The depth-dose distributions through the rat phantom along the radiation beamnon the lateral axis (side to side) are shown in Fig..3. These profiles indicate that the

TABLE 11

1 t)",, andi I ppcr andi Lowr Confidence Limits f-or Each Radiation Qualits

Electron 61.1 S6.6 66.4Brcinsstrahlung 81.2 71.8 87.4Gamnma 89.0) 7 .9) 97.1Neutron 98.0) 88.4 1()7.7

R %])Ii\ I]() \\D) N5(1 R Ill R I ()R\1 NM 345

z 99 T

-Bwrnssiahimnq

u 95-

90 - 'crr

z'T 80~

00

50-I 40~

S 30-

20Netr,

10

In

G amma

21 4 5 6 7 89 101214

RADIATION DOSE (Gy 10.

1( \C ICl.I I'd .JI\ lIII111 ICC: CCiCCI)1 11 11rrk'd domc rC'111I1xc titi Ic, ptodu-rd Ik 1hrii~th-

111,dT1C, .1it~ rriiiir IIc': O IN) 111,1 c11~ .dJt1fc - Ind Iww wnt'I Iicldk. I'\%~1-i~i 'jsr 'Nio1ill-

neuLtrons are attenuated I)\ about a f lactor of 2 From the entrance to tile csit side of*thei p hantom Ill hue II ' 'Co - ra' s a re at tenuLated b\ about 35' 1 . N V( eleeitrons andllrenisst tab i n \ ras s shoss a tairi'. Iat dose distribution throughI thle phlanltom. al-though21 thle elCtrIIons elIi hued a 20'' falloliin deposited enierg\ at the es it side.

i 1 ulk -; 4sioS~ tile dWose ariatioi a1 r 'si the uvcr:ea (p;t-ap Il bottom) ot therat phanitoml. and thle dlosc distrihutions Onl thle longitudinal aski, (nose to tail) areShIO\s n F igz. 5 TheIse fi&ures ind icate that thle d.ose iitributionS are tin i lorm1 Ir allsourI-cs. ss ith thle eCeCption l Othe electrons in Figi. 4. In1 th is hield, thle dose \\s s'greater at thle Underside ofthe phantom. i.e., thle rat gastrointestinal traIct.

tDtS( t Sl~tO\

I lie present data suggest that di llrent radiat ion tt n1al ities are not eli al l\ elkctix ein pro(iLuCi ng I P1). I hie ian k Order tor [P11) (least to most ellecti\0 e ''as neCi(Itoll. -i -

ra'. . brenCilsstrallIlIFIig antd electr-on radiat ion. II ighl-enerIgx elect ron's p~roduced [Ph1) atsignifieantl, lo,,ker (loses t hani tilie other fields. and] breCniStrahl ILI ng "s ra s pr-oduLced

pl1 a i iiihantl\ loss er doses than neuLtrons. Based onl traditional b)ologcl n

Poinits li ke tcll killing. miortalit\ . and cancer 11iduct ion, these Ii udinigs oppjose tilienolrm. since high-linear-energ'.-transter ( ITT) neutron radiation is generall\ consid-ered most effectis. and brernsstrahlung. electron. andi I radiation are consideredequalN (Aiketu C (I.-0. Since our findings oppose thle norm. %%ce interpret them as sug-gesting that radiation-induced ITI) may ins ol, e biological meehanisis difierentthan those ins oled in ell killing. niralit. and cancer induction.

1 2 3

DISANC c, FOM ID 'AIN SU

FI. e th d ' ro lc 1 o ,tle lt r l xsIlke JlIl )1 1 ie 1 1,1i11111 l 1 .11C2 ) s

"n m il/ d t, ll lli' ~w lstiep o ori n l 11 111 t haie fr m 10 ( .1- 0 4grcatr dt0

This is not th first paper t reor behaiora RB auslsha n o ih

I-F Iradiaions Fahl 111. Geoge c a~S~~l. 3 n 'lop n 1.1g4 eore ht-

1) 1 20

DITNccmFO SD AIGSUC

I-l. Icphds p~k ~fS h d~a si ic10mc 1 1 011idat~tCU0IIenooai~dtnulo fldI il c~rtoa 1100 o hl~etol IIll e.1. iz 4

llreatr dLO

This is nt the tirt paper t report ectioras ausls ta n bih

0 11 3 4

I~~ ~ ~ fo -3n exlectintroe ais

R l t t \t)' tl RP1 RI i)RSI N\ 1 341

U.1 TAS f ROM 19 S F

kitts thanT \ 'l-C iicutr~s I o ll unt I I I rcp( )rtCd that clcctrtt1ns l 1, dIsiiptd ra',t bhas~ [Ofat Itsc I Vc th .II I)k,1an 1as,.\ )t hc cc nt I IIIIIIII] f I~n a Ira cscar Ici 51(\% cd th Iat kCcct k Il

\\~cctcoi'c itlt~sr alaitin thcrap 111,11 than \ li AIII-'l\hcisrhhii id~U

\\if( !lc ho~c hchiax iltial Ittrk sli ccts ha IttG-l I I atI. ttll ar 01,11101) M.111.Cl C-

IikLt I (Ill rad latIt ill %,, as, Ilitic clickt Itk c tha.1n ktC -I t II anld 111 I I5 it I tic ct litlit )ncd

tast Ic a\ crIti pairadigiii li rats Itd tnrthI cr. th]at hidli-ciic I,-\ 1 I i 1ItIiki k-, i \\c scrc ck CTIiIII0 t'cf ctctis k., ( iid It ItIied ta I, Ic a kcrltt Is al 1"Itcha I IIIal .Ia rclatcd I to iianscaand1k \ unhitino1. (ITr tindlint' ar JcicrICIltiiliti COttM1 itli pict lowi ttIk. -Tt lch is

i1i1i 10r1a1it hckIausc It tsasitlc akcclcrttd Tat lmus)IC! as, a pui0tCiitia1lbtihtttc brTlaigc. nitrc cspciisi c hchdkora mod l.c.. inUikxi sd in thipst \lsO. thlcradiat itMolnl it\ \ ss rk suoIIgcsts that1 rad1ititn CitIsk1CIkY c ics ' isics a 'ctill to hC r-

)n daa littratosl I I') at 11ighl raldiatit i I l' hilt (111 1ns is0t NO Tiall anmanid ls Boctt,,, al I 21I reported that1 thL 1:1 )-, tO pruid nCC I PN i Il n [lklkc\ s I Isas

F .and it hias hkcn I Ill piled t hat the cIIh&cte Ic ima\ hc 3 tIN )\ . t, lIch micans" thcI P1 ) ctLo t CCI Ps nIcar tile I D- ) (6 ( 01 ' IT iN t hat TM t- con"idL -cd tile hestIAdlItio It oic Ilh1(401r TMfl. SC% cia! fact, ws mat acctiut )Ir thle dficrcncc.C 1 mrst1outask ss as i smiiti ltll)lll houm1h phcal lx dcnilandling tasks, arc Lcnicialkl ctmsmdcrcdliiiirc ra, ditiomis"iti CO)2~. \nlothcr reamon I- P1, is1 hIc inticrt maht, hC tile tmncltcstIcd alitcr c 5posu rc. Inl t hc mon kc\ ,tuoitk It). pcrtiirnicc tsas mninittored inimcdi -ac after CsposUrc 's hilc rats \kcrc teosted M( tmi from thic start oh irradiatiomi. ('in-pnhhishk-fcd rosdi sngssthat it rats arc tcstcd right alticm cskposnurc. tile I D_ X,, i11i uch loss, thani rcpt rtod here. \ n thicr rcason hiigh d isos arc neodcdI tot prid uco- ratFP!) ma% he simpix that tile rat is, radioresistant. s ilch is, conhirmcd for tile rat

K Ii il III Q T1,11 I I / I \ I' I'l I t fCII L'I I II

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hI 1ini .n c I c CI I " " I,%I II I \t\ I . .\ iN' -4 I \t\ I Inik nI Ic tI tI Iim I( I

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III,' Iiiinikcx 11i111iIi -I it \%JiN ,11 \1I (, 11 [C ,1% *. VI-c-TIi.'I!\ I)CI ITI TT Il l III i h ' ini nnmnkc

tNu l I t I,'l, I III\ I T) "t '11I Iii! I. na\' illn I I I5 \V Ii

I S) I. i liIIiccr t1an thatI of hc mlIonlkc % . . . an 1 0 0 reS pc ct I I\ I . InItil past. tile search for ti1w mcchan Isin of cfk'ct of, HI. o1)%as donic \\it h iln dcls thatdecnionsi ratc t1w effct. lo\o c\ r. anlothci ap'proach iighdt he to Stud\ nicl thatarc Ilorc rceSiStant. ice.. tile rat. or that do not dcmnsat [I.ic.. tile dog (h andtilw inonsIS I p lsw cscarCh)

Ill J-iiC\ 01N Insrdhiion LI naiit_\ rcscarn.h oin I P1 ).at \1I R RI. -CI IMsos o the Hi cqiaLi,1-tics inl radiation cfFCcti\cnlCSS %NCIC sUl'cestCLd. huit nol do1sHiIrill or tco' 'tical Cspl-

nat1ionl could c'splainl thce difil.ccs ?6). Ph\s ical factors poISihl\ nI oltcd in fihcdiflcring ScnlSIti,.tics to thle 1 0.1i 1_rdiation0s i11w,11C the a0ch0rac of N thedo C Incsure-mncii. thi krCnt211 malLcroscopic d1cpth-dosNC distibution01. ('ciccron dIoscpetbtisaIt ntrf 1s.dsc ratc Or- pu)lfsc timinl effect". an1d difk'r-cnt r-adiation1 (LUalit\ or- I I I

Sosinlctr\ ICCuracIo\ IS al con1ccrn Inl riation1,11 lualiZt\ studies fil A niim11hc-i.o oinwtIiC Icchii(uCS ar-c uNcd Ito '112SUrIlk theio deposited h\ diflerentI riaIons.11F or hio-cncri. ''lcctrons arid \ ta\ s. li oh dows ratcs ii tilhc hcain pLiNC (sc I IN Ihl II-CLtIIi\'do tile dL'tcrIlilntiOnl and( app111itionl of' 1II/ationl satura' tionl orr1cilons" toe aT:Ich ha IinIhecr rc adin I n L ( thc c omnIpIicaon in I I N \I(s d os nSir 111 c I d' - i I Ir leI-I1g"topping poscr-s of leCtron01s aid hremIIsstrahluno \ ra IS conip1arcdi to CO, and~ tilec

ioniiation-chnibcr-spccifi corrcction nc d d to \'all1 effects and disp),lacIenof' phantom mlatcrial 1 i tle chamlhcr 1 22). Propcr handling of' these \ ariahlcs IS as,-Surcd at AFRRI h\ colkihoratini117 %kith I!c NaZtionadl n1SitUtc of'SItandads anld I CCli-nolo ig\hrincrl\ thc National 13u-reau1 ol'Sta ndardS) inl routinte ion i/at ion chaiihcrcalibrationl and Iricnkc dosiITnctr\ si udics (23). Inl k'.l it icI CCura-.CI \\ZI as nailn-tai ncd for tile non reactor radiation, for oxer a dccade. Si ncc nlo r-cfcrcncc c ists I'm

neutron radiation. a , -I 0'' standz.rdiation is achicved at AIR RI h\ adherece to

R %\11tt)\ \\D) \ir(Tr III Ri "R11\\ I 4i

spIcci t ic pirotocol 2 -4 1 and I Ir ItCrcoIll pai''sons bct\% Ccn d II tlcrci tc I C i t l ILI c. a ndc :abo-raitorics 0, 2 4 1.

I trr1t1hcr elf I Ir1 to idl ist , d thIl d It cr'Criccs in1 bchas \oral scsI tIs% it ito raldiat[inkqt.1a111t l s IcrC,' i I'Casrilc 1)t" ot, Z~c nuicroswcopic d'.pth1 --dosc, distrrtrI II oll ( I lus. -'.

IW1 I tr 'Il Mhrs tha 0111dosc dcpo'Itced O\ cr Ih IlenrIth 1 of Ih a1 "t 1i tWillrn. .. )hNO til, 5" ot trmc 'I thdiitions Inl I W, anHd -4. 1 IViTC3 slirrss, thatI 11cutr1Ontell oil niar kcdI\ o\ er tile \\ dth ol the raS. surmctiric that rICUtrons1 \%cr less Ictlrct CI(L\C,I LC to 11 I Iil rnIIIt\ onlI l CM cI side. 11 CC Iocc., NC\ca thrii ar1tic LISrrs thLUCJ J Isl

1~it 11irs1. 111I11t-l1 II ilel.troris1 Are riorniall\Is pcri .tki.1 ciihcs\ are riiorccl c cInl prodIrICI I uc IL! iloccl CtlCCIt like cmUCSis. MOralits1 ar11d c'anlccr tiurnl hiss-

I I iataIori. 'Scc r Id. t II ICI)" cprItc Cdic It Crc ~ rtrc at 1C.'1k11 tile rat rI IIdIllnC. 1I htrs.1s 11rC Ilc ntrIIh)cr 1 oleicirirori LciCO td on1 tlic C\it ic \%I, as css. it ss, asrcatcr onl

IlI C CntranlkC S idc. I nal I I% Your n ( 14 lconIl uLctcd a1 ,t rId\ Col cI IV Ia Ic tI c AI,,t ot,h VC M,1-IsIt I ll I I- \ I'T ,,Iand 11CLIro rad11iah'tion onl Ilon kc\ pcrtOrrnant.c Ill %\ ic~:lh cdctJ s c radcn 0)as co I \ 1 nstan. IllIiat stIud>. thlc I D_> for \ ray %%,is ,I gi IIIcals Ilosscr. than \\ ith rICLl ori ra.diatrori. I hrs C\ rdcnICc stri_1Cccts, that tilc rrctrr-orilllo111 ll rd~cposiicd dosc Inl otrr Sttrd\ Is prOhahls\ not tile Icasori) IImcttor radiation1 \%,Is lcIs

\s ltrrthicr shiossil Inl I :it. S. CII clcct rondo cItcosi1t o dcICrcasd across, tile lateral,1\1s aIhofrt 2(0', . anld it irIcrea'Zsed thc sailmc onl til hcsrtical as s (top to bottomll I asshloss in I Fig. 4. 11 b i ncrease inl- [i.NI s s ttrf)ibute to clcctrtn scattcrin [i rom thcssood platform b)closs tilc plastic holdcr 191. 1 losseser. it is ditlicult tor asIt'a 21',r-cUrona1 lnon liii trrrr ', s i d lter1 III ra d itirri Cfctk'ci css. kIor i nsta ncc. s' c reported

a no riiitoril tinleh bremsstrah111 I- ing iLd. ice.. aI 2(0', drop \\as nIOtcd inidhcad (6).\hcam tlattcllr t orrectcd tilc tallolfzind thc c1 Ictis cncss oI thc X rasN sas tested inl

r~ti ri i tPrml held. I li nic"s II),, did not di ter from t hc trigi nal It u1.s. ss Itilc correct-rig1 ItIIlrI Irit'rr Wit I i Jstliecd. I is CItICStioriahle .\hethcr til c (1 rronal 5 ariatiOnls

noted hcrc can aIccori1 fotrk t he oscral I diftferences Ii radiation ctlkccti ecss.,,,Ih ICII ha nccd ctItisccsofcet ons1r IP!) n1uts be (I ric to dOsc i nhorntrccnc-

Ineis of" I ih-cricrt\ cci runs s licre anatom icall structures rltCrt"ICC. I eCtrOn dOscdistr~iIOI btt1n at itetUccs, d iSpla> coldI Spots I lcss' dtrse Iin dcnscr matcrial arnd hotspots ( highcr dorsc I inl less dIcIsc material (26). [or 10I- to 31-\c\ clectrons thc si/cof, tice ctl'ccts cmn he t15-301' at siminple ai r-s atcr i ntertktccs and IhltS as i ich at boric-ss ater interlacWes. ss ichl can externd o, erazrcas tintone to scral millimeters. [h le mag-nitILd urfc !1rt , Il hcsciii oinngecli tics is; amplified at corlIples ariatoinlical StrtILrcr. likc

01 hOL. Ili iCU- tcatrr hc ma ZbI t'ZtCtor in1) IIfucc16i tte c tieCct cagdprihationl on tissuIcs sti rrou nldcd h\ bonc. ice. thle spi nal cord and til h rain, as ssAll

rrOf ot0los tisSLIc derTISIts ic.. thc ling. I1 clihargcd-part ce dosc diq1ribuI [o(ilI, propriaLte phlanttrms is ;cqrI i rcd to dclin tilth potentialI role ot' local i/cd (Joise

'trinecicitics, Ilie state ot' tilie art Ii electronl bcarrn dosi met r has ads anccd sot[- .rcaihstic clculatitirs nut>\ riti% he hpossiblclfl

A\ notheir reason that tlw (Ii tkrnt rad iationis s crc not cq~ualI> cth'ct isc onl bchaso iorina' bc dlose raitc. ( 1corgc a/ al, (2S se d rcactor -, ray s li sIho\s anl 804 l-\~ drop ( 100(hr 2. I (s(nhcLIotpgerritrc rdscatcs of, > to 210 ( 10 un1i. A notherstutId> reported that tsk o split 4-t-( i electron dloscs crcated a 1 -1 in tiruic coLrrSCfor repa ir ar( rdocclropmnnt of' i in in iII it> to degradationi from1 tile second dlose inl

35~ UiO(, I %ISt\%. \\p ) 1)()I1 I)

flh 'br )CrTOI 11rmncc (29V . BIr Mier (Is') Stated that I'DP1 i ncreased froml -' to 8 I at -

dose rate,, 4t0 3 to I .S G\ 1111 ml n inmonkcs s gi\eci a total dlose ot' 10( ( ;s. Ihewc Studiessulgest that a conip1 lc \ dosc-tinmc doc- ra tc relatiship cs ists tor th hc bhas oralctlccts, oftradiationl. Inl our1 Studics. tile pulsed nature of' I]N,\ irradiations, I lable 1)Is dittcrcni t f rom I tie Conit inuLous C\posurIc VW1it tice "'C oSource or thti rcac' r. withthlt clcc tro n bcanil ha% Ing a f1Iit tod loss Cr puilse rate thani tice br1cnilSt rah Li rg \

rs.Not olsl Is tilic I (\A( *dosc dclscrs, parItior101Cd Into riicroscornd pultses atratec, of OCt cal- bil huth cactiI IiNsc di\5 dcd ito a st ructuLire of' radiof'rcq iUcncs ss as c

packets.l Ii us t tice I IN V" radiat ion timeii scales arc Such as to p)OSNiblI s iiiipIicatc sce%-cral lc\ clS of pti\ sicocncricat iicchl iism in tl tic dosc-tinmc dosc-ratc rclatisih ips.

[tlic ph1S]i sIllmcliiss sshiicli miigtit he implicated sstin a, biological effect dce-

LcaSCS \\11 Incr-caSi n IA I Call he cI ULActd b\ conrsideri ng thti characcistw i 0socctan Ill so IdL-Statc d osiiictc rs. Iin niari\ I)ti\ s Icat Is\ Stc rim tlic rcspon SC to ricutron rad I-at ion is k rioss i to bc lcss than tilic rcspOiSC to CclLii salcut -ra\ doscs. I -or c sam plc.Inll ItIIId ioni/ation chariihcr1 (-4)), It IS tice initial rccorniination of closch, spacedItn TII /atio0ns prWod uccdi b\ t iiti- I I I radiations "s ti Icti causcs nicutrori- Ind uLccd ion /a-tiori Cuirrcent', to bc Miiuchi los cr than thtosc prod uccd h\ ccfIral doscs of' , ra\ s. Inphotog 'ra pIc t iIli 1(3/1). M5 0 phi\ SiCal Ictlects Causc a reduced selils It It 'r h igh- I-FF

radatiii coiiariclto losI.I radiations aS tollWASY (a) tlcdcrisc rorir/ations alonga tiiet-11 I particle track arc tar In csccss oftliosc niccdcd to Producc niiasirnal (feli-Sit%, and iucli oft tic dcpositcd cnriere is w\asted ( ox cr-kill ctlhect"1: lbh1 tlic cascadc of'scconidarx\ CI~rl p~rodUCCd bs \ and -, ra\ s Causcs loss -I T~ I ariplification of' ticenumibcr of'photograph ic grai s produiccd b\ cacti 1Ci I'tpotl.Illll.IlTDtile rcspoisc'( \ is, rcduccd M icri tice rangc of particutatc radiiationi is lcss than ticedrariictcr of tlic II [) grains: this srtc-sic-spcrtic cffcct has hccri attributed to aninscrisitisC coutcr laxcr of ttic I [,D grain 132). [tlic cxistcncc of thcsc phvsical effectssuggcsts that tlic bchas itiral ctkects of' ioiini raito I ia' haccpicable CairS-at i c rIicchian isms tilt a Sotlid-state ph s scs or ph sicochicnicat lcvcl. i .c.. ionlic or radi-cat Interaction \% itti ncurons or ril imbra ncs. rathicr than v\ithin the traditional micro-biological or biocicrical thtiorics.

IIlc Currceit data nia\ also has c implications for tic nianneid spacc program. si ncctilc rclat is c -onipariSOriS Su~gg-cst that tirgh-cncrgx electrons (common in space) (33)disruLpt bclas ior at losscr doses than othcr radiations. regardtcss of ilic (loscs neededto producc [P1). [hIis concern nav bc grcatcr nowv than beforc. Siricc future probesss ill hc dccpcr inl spiacc foir torigcr durations. oir tlc spacecraft will use geostationarvorbits s itli lcss atnmosphicric stuelding (34. 35). .Also. mtiilc riassive radiation is notnormial in space. Itirlk high lcscls have been reported fromn largc solar proitoni cventsI 33). For inistancc. inl .*ugust 1 972. a large eent oiccurredl that w~ould tiascgiven spacepcrsonnril 2.9 (6\/li (1r I15 h for a total skin dose of 44 (is inr a niorrial orbit. Al-though this cs crit w~as thle ssorst repoirted, the Stud\ (if solar actixit% is ricw and thess orst casc iax bc u~nknown. Other space radiations niax also atlect tlic bceiavioralabiIi t\ of'pcrsonncl. Such as high / and high-cnergv (I ZEt particles that can ceatcmicroscopic lcsions in tissuc. Reccnt %sork showed that "Feccan Hinidic a conditionedtastc ascrsion (a variarit if naulsea (3t0lf and disrupt miotoir pertforniance at ser\ low%doses compared to ), photons or high-energy electrons (.?. 3M). Iltlis is true, a stud\of the effects of space radiation. "specially, IZE particles and proiton radiation, isneeded before astronauts are a regular part ouf that ensvironment.

R NI \11 WI\ \\D) \1((l IIR 1 RI ()R%1 \\( 1351

V( K\s(AI I IX I I\ NI s

Ihc .iilhi, thanik ( Nl)'.ard and ( I( IKCNIssell IIII ICIII Inhi~a sstitcIttraIningII andI testiungthe LII [ uIf' Is. Is 1J klSiIn 1 Ilisstil it.,liN 'd is. anld R \k) u 1 , it i )ielp Ity I, I ri tiltte the researchi11I h' -eICT, 11 \a'.spitd I'\ Ow- Ni tid I itc' RadiohioilogN Reseacikh IInitute. DCIW )e \eNuLClar

\ "1,% i) \i i LIderI RVsiCtch 11 k 1 1 111( NI.) l3It'tt. e s pieselNIe D IItIns 11mpei irc those It fieit tII I,10Crit, I,I tIC II II I I\DN N 1 11a' fleet1 elt l s1 t 11II Iidl h Iii tiit' kd

RI i l \ I n: '\UgUSI 11). 1988: vN((I III ID: Jartuar\ 6. 1989

RI I I Ri V( I S

I A I) i' I ol I )c Ihts % rl t , 1\ 1 t \ Ilfi U Ced h ' i ct IC I IInl/ I Iit rid IatiIn I 'ot)item7, .' I k , ' 1! 1-0 Y. II)'

N I, ( ( , It R\/ anId R. s'il N ( i I Ileels Ai radiationj oin niinlkc\ isual di',cri iniiiatioii

pei tOrnian, I, n I R,i,, oPi R,,ii it I., NI. I ieldcn- J . I ,Iklut . JI I I Ilendr\ .and I ) Scott. I ds.)%d ,II p. 2 5', I ,,slii & I ran,:ts, I iidtn 11>7 [-\hstratIj

R I (a if tl 1 R. I.,(' I N ip I . I ). M. N HIl R It1. and 1 . I . B \R R( )\. I tic rclattsec ctlectis eness, of

ti'st it nctie tns for muinitatuitre pig perfoirmiance decre ment. Rodiw Rs'.' 4K. 3 32- 14 i ') 1' 11,

I4 k1 Is lif( til and R. N Y ii t. Niti \*it'I /I//(11101''tc' II' ( ivoltlIn, /11, ItohA" it\ I'i/,t Scienl-titte Repotir SR "2. Nrined [ orces Raditittltig\ Research Institute. Bethe',da. N IlI). I )- "

13N lI t . ( oiiiiparat i\ c etleets iii brent st ralil ung. ga iiima. and elect ron raditat ion in rat niotoir per ir-

malice l it I , i''/tc It I/t, 9117t S it/ ' i it ofM0/P I' I tI It t/n' OW 017zoiIi'itt "t /)C't,'t wi. pp. 68-72t nitcd State'. -ir I tirce .cadeis . 1984,B4 K RI tt t I let'.s Itf hrenis'.tra h U nti anrd electron radiartin tin rat inoto r perfi ifia ne Raial Rt''

IN, ', I - 21) 11 9,S4),BN lx it a I V I\ I if I .and R . s'rik tN (. (I itnpan 'on t'accelcrod anrd rota rod se n'.itti\ it\ in detectintgciba no I- aid .tc r\ lain ide-intdlICed ft prtirma neC deC(reiIenI in rats: Re\. en% ol-es penrnicn ia co nsider-ations. ,itrotating rod ,\,,tcins. ,'ttoto/t 2, 761-787 ( 1911.

an .1 3. ( Nit D4 ), \i St I. In terct iipar'., n tit' neutron dotimetrs technique'. at the \ I >RRI I RIG(r-C.c,ir. R<adia l lo~tt 23. 317-312( ( I M8).

-itt Oam o ttti t- antd Ri a, iot irad(<tn/tat tin/tC/t/ I ech niCal Repoirt 86-5, NArnied IFrce' R ad obi-iliigN IReSearch intitute. Bcthc'.da. NIDI. 1986.

it)%" s.1 I)\ and I. .1. NI VsM N . JR.. /itdiitn tutu t ttia/ .Int k Nle( irass -1 ill. Ncss ) ork,

1I I I.J . I ],i\)It Quanta) rcspti usesanrd t lie ttleranee lt distri hut tin. In Itat sta 1itt tst it I/iI e,

I ''Itt. pp.3;4

()- ;61). NMacnmillan Co.. Ness )(irk. 19711.

R I Rf Ottttittht'C( 1,ItIN 'I/ittitl I),,,iiiitit Rttt/tii/mni/it Report 31). 1internatitinal Com missionon Radiattion ( nit'.and Measurements, Washinglton. D(X1979.

3I.? WRI7'. kRtdhatw iAl~,toetrt :i Oernt em ii1

i'Ottl/ lcn~i i if Itmi/ 15)) Aft'I Repotir 5.Inter-

national( I.inissioir tn Radiaion I nit'.and Measurenments, Bethesa. Ml). 1984.

apidt Sl'tttllt/ it/ 'mtitit IInisersit), Microflilm InternationalAnn Asrbor. NII. 1979.

Ii' III A. fit \i.( inliparatise etlects t)ifXPtire tthigh-energy electrons and gamma radiatioin tin active

as uidanee bchas thur. bitt .1 Rt/tt I/au 44, 257_26(11913f.It, If 1 I. n ts. 13. I \of~i I NI \. and I). ('s-i'. Ratitihiological eflecliseniess I 1ill' meigasoltage V-

ras and clectrin hearis ii radititheraps . Rttln Re's 1015,58-67(1I986).F B. NI. R\RsIN. . . Iii t -iand 3. -N. Jtisft if.An as ~ssnienItifthe hhas itiral to(tsiis t'higlt-energv

riot particles, coimpared ii) tither qualities of'radiation. in press.I \ N it 1111 R.. liitrdate di us-rate: eflktv sf til'(I it i perl'omaiie anrd blootd pre~'u min fi moke s.

Radi Ri'' 70, 1-8- 114) (107/I) \ I' C'. il i i . Ril/it 1iui/uet.! Irentifee- 11all. 1 lnglessotid Cliff". NJ. 968.,o ( IN NI( it) .1 I I '. ittitand B. Wti. Behas itral and neurtiph'.sitiltgical clianges associated %k ith

c li i,,retilifi ingra tti i - lIes/l i i l l tl rt I It/i ittw1R . I.WS alkcis . I . . ('er en'. -and

.3 V % it luesen. I ds.i. Viol. I . Part 2. ItIS. Arm\.- Washingttin 1)., in pres,,.

'Is I

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IV IIimrm.aind 1) ""Cilt. I d' N At I p. ii. I a'lir & I ranais. 1 01i1,l0111, 11)117 'lrat

AAMEO F0RCeS RA01IIAOLOGY'ESEARCH iNSTITvlE

SCIENTIFIC REPORT

ii51! 1i'\I("ISk ii ',Li43 liSiiSR89-1 6

Gamma Radiation Affects Active Electrolyte Transportby Rabbit Ileum

11. Correlation of Alanine and Theophylline Response with Morphology

Px \ \I I \ J. ( 0 N I I R-S.SI1I I I

I I R-Svi I ii. I ( ianiniat Radiation A11 ects \ctise [ lectrolsi I ransport b\ Rabbit 11-CiUM 11 ( orrclati0n "I \LMiIC andi I heophs ine Response \kith NI rphilogs Raialu Rc'W1. P) 4 " ( S'it

I lhe tcspoi ot H cal N"eme nt 1%Is i LaI fromii rabbits to an acti el\ t ransported a niino acid and.1 sCctanoLC, "Jc s s al ua tcd filoh i~Ing c 5posa re ito) (i s sshole-hod s -,. Irradiation I he abihIt

oIilal Ncgim t respin jito thc acti i tiransported aiino acid, ali tmie. \% as not signilica nt,1i1i11nishWkd until 916 Ii potsosr ecrcased responsis cness to thc scretagOgue., theophs linc.ccu-Lird cathrliat '2 11. 1 lics.c cfILMects r11ot appcar to he accounted firh h% decreasedo food intake

Al irradiated allinmals dlonc I \anmnation (it Intestinal morphological change% ib respect tothe hcsha nvcN it elcttril Ic Iran spi i ret catled thit dec'reased anl'1 n acid transport ciii nCCidSLsitlo hss ot Intestinal s li. \lhoughI a miorphlogical correlate ot decreased secretor\ responsexkjis lii ' as stik in as that fo r absorption, the theiiphs ili response appeared tiidecline coticlni-iat ssith lthe .ippearankc o Hincrcascd miiti tic acti t in t hc Intestinal crs pts,. I he resultsof this

studs ' Indicate that. tolios i ng a dose of 1ii (i. the inabilits oif these tisusto respond to amitioaci' usIs dL toi a Ii ss 1inat UrC silu absiirpi se CellIs subseq UC It to den Udat ion oft he I ntestinhalmiuc isa I here appeared iti be little Imnipa irment iii cell tiieni brate Ira it purt processes for ala-nine. In contrast. the decreased Necretor\ response could not be correlated ssith the disappear-aMice ot am ne t cell Is te antd perhaps, results friiti intc reased pro l iferati on in the cr5 tt at the

IN I ROIt V I I0N

-\Ithouuh ctlixts, oft'oniiing radiation on the gastrointestinal tract have been welldocurnented I rc\ essedc tn 1/. 211. recent studies continue to pro\vide ne\% insight intothe miechatisiss U ndcrl \i ng post Irradiatiotn dM'Lu nction. Man\ Studies have to0Cusedion thcefftls of radiat ion on nutrient absorption ( 3-S'). Vhe1re is. however. essential]\little inftormation concerning the deffiet otradiation on the other mode ot transportin these 1tissies. electrol\ c secretion. \,ot onl\ are Cellular transpotrt processes under-l\ ing secretiotn dilkcrent f-rm those associated wkith absorption. ahsorptiv e and secre-tor\, cellsappear to he locali/ed to distinct regions ofthel Intestinal mu1.cosa. \illus andcr\ pt. rcspectixecl\ (9-//). Recent ohsersations trorn this laboratory 1121 indicatedthat aetis e transcell ular electrol\ te secretion IS Stim Ulated 24 h Ibillovi ng radiationesposure. Il hich mas" contribhite tol fluid and elctrolyte loss. I hus iniljirmation con-

419 00; 1n -iX7/X') u00

42() P \1 hI A J. G L'NTF2 R-SNI I TI

ccrning the ctflict ol radiati on on secretor\ as w ell as absorptixe processes is importantto an understanding of intestinal dysfunction post irrad iation.

To examine the role of membrane transport processes in postirradiation intestinalds sfun ction in greater detail. this stud\ assesses in rabbit ileum the eflect of radiationon tw.o well-characterized active transport processes. Both absorption, stimulated b\amino acids. and secretion, stimulated by theophylline. are evaluated. These data arecorrelated wkith changes in morphology to examine the relation between functionaland morphological damage induced bexposure to ionizing radiation. In addition.since absorption and secretion are localized to diffe~rent areas. the results are discussedxxNith respect to regional ettects of radiation. Trhe results showk that both absorptionand secretion are inhibited b radiation exposure w-ith diffe~rent time courses. Further.in some cases, this loss of function can be attributed to the demise of a population ofintestinal cells.

%I E IHOD)S

MIale Ness Zeailand W'shite rabbits, Olaclton tDutchland. t)enser. PA-\kcsighing 2-3 kg ssere screened b'rOs dce~c oldisease prior ito use. [he% Aere individuall\ housed in stainless steel cages and maintained mnrooms ,at 21VN. ;0t,, R1I 11 light 12 li dark (no twilight). The\ \%ere allossed access ito comnmercial chossand tap ssater oct ihaiioi ececpt f'or "f-asted"animals from ss hich f'ood was ss tthheld. All procedures usedfor aninmal irradiatioin and assessment of' iransepitheltal transport has e been reported pres jousl\ 0/2).BricIl%. the terminal ileum %sas isolated from euthanatjized New Zealand Wvhite rabbits (811 mg/kg is. pento-barbtal sodium) that ssere either exposed to tO (i sshole-bods "Co radiation, sham irradiated. or fasted.I[hes t issues are refe'rred to as irradiated, control, and fasted. respecisel\. the tolloss ing times "sere se-lCted 10r Isolation oftiUssues from the animal: 24. 48. 721. or 96 h postirradiation or postfist.

Ifor the assessment of transepithelial electrical parameters, the tissues \%ere mounted in chambers andbathed %kith a standard Ringer's solution (12. 1Iransepithelial potential (P), resistance (RI). and short-circuit current (/.,. ss hich represents the sum if all aclise transcellular ion mosements across the tissue)ssere monitored as presiciusl% described (121. After steads-state baseline salues %\ere achiesed Igenerall\Sit- 100 i m. the response of the intestinal segments to an aetisel\ transported amino acid %. .sdetermined

hthe add itioni of alan ine from stock solut ions ito the u m inal bath (fi nal concen t raticon. 10) m .11). Subse-LtucntlII. alanine ssas, rcisoed allowing the reestablishment ol'baseline salues and then replaced by Ringer'scccnt~ucning the secretagogueLI theoph~ line ( 10 mI/ 1. the response of irradiated and f'asted segments \'asalsxxcs comiparedc to that of a control animal from the same "batch" of animals to minimi/e differences-arising from ditlerent groups oft animals, All results are expressed as the mean SENI. Significant differ-Clnces Iron c01ontrol \\ere determined at the 1)1)5 lesel using the Student's ttest.

Vdlitional seg!ments of tissue isolated from the same animals used in transport studies \%ere preparedfnc 11, rphiclccgicl exaninatcn. SectionsA sere stained with hematox-Oln and eiisin and examined for quali-

tiic hanges, in mccrpholog\ pcistirradiatimn.

RYSI ITS

I ri1sftoc II studics The results ofa ty pical experiment are shown in Fig. I in whichthe response of ileal segments isolated from an irradiated animal 24 h postexposureis cimpared to that from a control animal. Although the baseline 1,~ was elevated 24h psistirradiation. the response of the tissue to both alanine and theophvlline wasessentialls identical to control. Previous studies from this laboratorx. (12) have shownthat the elexated basal 1., postirradiation reflects a stimulation of cellular secretoryprocesses (blood to lumen) that are similar to those elicited by secretagogues such asthieophxlfine (/0). '[his diders from the ionic basis of' the increase in /,. followingalanine addition, wkhich reflects increased transcellular absorptive processes (9).

11I F\ \BSOR IIIIONA AD SFCRFIION POS IIRRADIA 110N 421

200

1 60-

-120-

80 f~ Irradlation

40

0 100 200 300 400Time (min)

I 1(. 1 , 1t ofical segments tron a control and an irradiated animal fI 10 (i\ I ssith respect to time attermou0Lnting! in chamibers. I ach point is the mean ol salues tbor four segments from the same animal. Arrowslabeled 1. 2. and 3 indicate alanine addition. alanine: remos al. anti theophx liine additioni. respectivels.

I-ie time course of' differences in the response of' irradiated segments ( 10 Gy) toalanine or theophvline with respect to controls is shown in Fig. 2. There were nosignificant differences observed in the response to alanine until 96 h postexposure bvwhich time the respotnse was greatly diminished. Significant declines in the responseof irradiated segments to theophvlline were observed to occur earlier at 72 h postirra-diation.

Since food intake of'irradiated animals initiallv decreases fol1lowing exposure. theeffect ot tasting on the response to alanine and theophylline was evaluated in control

20

E 0 T

-20

-40 -

40CL -60

Cr -80* *

44 -100 *

24 48 72 96

Time Postirradiation (hours)

Hi.. 2. Response of irradiated tissNues to alanine (hatched bars) and theophylline (tilled hars) comparedto controls with respect to time piostirradiiation. AResponse A/, (irradiated) -%/, (control): n - 4irradiated and 4 control tissues (breach bar:. indicates significant difference.

4 22 1 \%1 H J. AJt N I [R-SMI [ff

I A\! 1 I

I he F l .ct of I asti ng .:" .he Respon- eto Alanin, and I heoph. iine

241h 4It /h 2tJ 9( ol

l.anlnc 6 .4 ;4.8 , I .3 17.7 2' 22.3 . l.11 VS 0 03 NS NS

Iheoph ,iio, 1 1 2 • 144 28_) - 57 42,9 ' 210.6 44.9 - 20.2' NS 1)3 NS N S

,,, Dalaar c c\prcssed asthIe nican of A (.Itasted) A 1, Icontrol) fbr I r e pen crl ns. N.S nnsig-nillicunt.

animals. As shown in Table 1. the response of"lasted" animals to both alanine andtheophxHline was decreased compared to nonlasted controls at 48 h poslfast. )itllr-ences in the response sere not significant at the other times studied including thoseat % hiloi signilicant ctects ot radiation were noted. Direct compar!, .n ,' t1he ,.,:ingwith the irradiated data (fable I and Fig. 2). however, did not show significant ditthr-ences between these two groups at 72 or 96 h.

Ior,hohi,,v'. The morphology ot ileal segments isolated irom tissues used in thetransport studies above was examined in an attempt to further define the factors un-deriving the changes in transport properties lOllowing radiation exposure. Figure 3shows the typical appearance of an ileal segment from a control animal. Long finger-like projections. villi. protrude into the lumen with numerous crypts at their base(Fig. 3A). Cells lining the villi are columnar epithelial cells: their brush borders (com-posed of microvilli on the individual cells) are clearly visible in Fig. 3B. A smallerpopulation of mucus-secreting goblet cells is also present. In contrast to the villusarea, the crypt area (shown in Fig. 3C) has at least five main cell types (14). Three ofthese are easily identified in the field: Paneth cells located at the base of the crypt.goblet cells, and a proliierative cell in which a mitotic figure is visible. Argentatfineand undittirentiated crypt cells are not easily identified in this section.

The eltects of radiation on the morphology of rabbit ileum was similar to thatobserved in this and other species for a dose that is considered "threshold" for gutinjury (1.5). The severity of the damage was quantitatively less than that associatedwith pure gut death and a proliferative burst was observed in the crypts at the latertimes. The time co,,rse of the changes in morphology is detailed in Figs. 4-7. At 24 hpostexposure little change in the gross morphology of the tissue is visible (Fig. 4A).There is no obvious blunting of the villi and the brush border membranes are stillprevalent (Fig. 4B). There is no major disruption of the crypt epithelium, althoughdiscrete areas of cell death are visible (Fig. 4('). While the radiosensitive cells cannotbe identified in the figure. the Paneth cell population appears virtually unafliected.Interestingly, mitotic figures are not frequently seen at this time, suggesting that, whilemajor alterations in morphology have not occurred. mitosis has been interrupted.Blunting of the villi is observed by 48 h postirradiation, and a greater proportion ofthe villus appears occupied by goblet cells compared to control (Figs. 5A, 5B). Celldeath in the crypts appears compaiable to that at 24 h. However, at this time n.t,

It FAL ABSORPTION AND SECRETION POSTIRRADIATION 423

CEC

GCC

t(;. 3. (A) Morpholog\ of ilea] segment from a control animal. Villus (V) and crypt (C) regions aredesignated. Boxes indicate regions from which higher magnifications ((B) and (Co) were taken. (B) Highermagnification Of vilsshowing goblet ((') and epithelial cells (ECI. (C( Higher magnification o~fcrvptshowing goblet (60C and Paneth cells (PC). A mitotic figure is shown at M. T-he scale shown i~s 2(X) pm.

424 PANIFLA J. GIJNTER-SMITH

2

~4 low,

21

'II -E

~-3

ILTM.\ \BSORPTION AND SECRETION POSFIRRADIAII1ON 425

ciao

42 RAWIII A J (J'N I I-R-SNII III

AA

IRI

iB !ci

4'

Ilk

11.6 ( (ross rmorpholog,. ofleIal segment 72 h postiriadiation. Ifigher magnification ot %illts andcr5 pt region% in (B1) anrd (( - is taken from ditleren t sections but from the same aniima . A niti it tigo re osshmk n at Ni Scale is 2'(K) urn

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'WELOW"'M

tietUles ate tbser\ ed Olite 5cii C ). Simi1,1 larealics III imiorpliitlltc are' ith11t ed 12 i ITs rad Iatio I ik s )I 1 0 Hg.I V 11 i unIIti ile (11 tile \ 1111 10 Ct t to h cIn-

cresed IT iit IlibtO Il tit Iti tOI cure 11[)pCanncI in l thle ers\ pt aIea a liso) seemsl to) 1a1 cIcid iC nolI[ I iL ( B\ 1)()1 Ii hosiriattI ..0u1nt1inh 11Il tll f \ I 11 Ixis prteese unIttl

t11c\ tic \ itttAll\ abseit 0 w,: - \T \\ here no0t denuded, the: \Inls rentilnts are CitS -

Ci ed ws ill etibontal lathet-It c,1thlunitar cclks iF i. -11). 1 lie oserall depth oftthcer p:-IteCtelin ha) IIIICIIITTInIIe Teased\kI. MlItok tiIectienres I r numerous 1111 W11' and Ltsr 10 ehe 'I]A (I the tic:kcI nI Oft Te e\ pt, 11, its 'I"s nott seenI 1t ear1lier tImeIS. I hLns thle entireC ITnLC~sa appe)arsN

be1 1CI ITi t 011- hrtI Ililt11CeMr \Ctill uSt at this, time.c petrhaps) Ii an1 attempt It)I repIpn)IlattthW ITitic'a \\H1 \l 1.11 a ,l cells.

1 1155 "l I~t\

Ill an) ear11i st11)(k f> chanllcs Inl basal trajl-iseehluhar ecls tk( traiisport pr('(-Aese Whhabbt 1Cisn olssIl1k et esrbesith respect to dise and1L timeI a1ICr r'aiaton

eoslire I li resolt in~dlicd that1 a sItiniialatntn0 ot electru01t te sCre-tors\ pr[otcssOccurredC 5 '41 1)1,010,111 potraitoita ssicse otinct i ad eleCktr-1It It)los assoited ss ith the eastriitteIstInal s\ nldrontec. Ini the present stud\ change,,Ill thec ph\ sthi0,CAl 11n nett011 o) the Ilitsinal nlucos"a ss ith radiation hat e beeni C\-pli re d i1 tt treatet detl an ld c-Olrelated \% ith Iorphooical chancs. I he resp (11w ot,Oeal seenients N-0in1 ITiT-, d o animal uas deter11mied J"01 )"oth an) aCis\J ci tans-portedai no cid. a laine1. and a seCre'tag0ogueI. thlCophrl linte. SI ice thle resplonses ott1h tiu Ito these aei-lts hasC he~ bci I rs oiIS\ atibnhted tOditIkrent cell Jpilatioiis

11 C\/ 11.al11t0on 01' theseI proteeC se aloss itlciel C e\;tliiCV111ila1tioii Of tuLnctionaldanIIaC tot the"se tss 0 rii'ls" it thle Intestinal mu11COsa.

In1crea* ses 111 /,, hilloss inc1 the addition otactis el transported ami111 nit acids and sucg-ar to,1k thle In0u.tin111 sitlstiut has CILII eCI prsos enShkIos n t retlect inIcreaNed transllnIL-Ia Na ab\d, torption (Y). NaI absorp1l'tiOn is stmltdbcause thle iinU\stlucars and(amino11 acid" Into the .cll Pisergeticalllr coupCld to Na Clitr-\ Inl additiOn. os rallIlin i I)bs) rpt It ITT I s IIncreased In IT Ilie process dILe to t01he resulIt In osmoti11 (11c dri In ng foiree.Pres ions studies has e prosided comlpelling es iIdenee th~at Oink the COILum ai)thllialeelk Isfiling the inteinlal \ ill i acquire transport processes req i lred to) ab1sorb1 sugar anda Ill Ii() ac"ids at si()Tile time1 dur-1ig Ill igratIin alIong thle \ ill us( 10t). 1 hti the11 assessmen~tit thle response to alaine tOilloss I ng radiation cx pOSiire speei healkl probes one facet

iftihe Fun~ctional lintegrilt of'tI li TiHUS Cpieliuml.\ sign ifican t etfect ott radiation onl the response of' teal segmecnts, to ailan inc X\ as not

)itbser\ edI until % h p)ostexposure~i h\ ss hich timec the responMse ss as \ ituaLllx absent.Miorphioliogical eaiim ation ot" the tissue Indicated that tis decline ill thle responsetit alan iec ciincided %%~I th the disaippearanee of Ithle intestinal % ill i Si ne the electricalresistance itt the tissue 55as pres, iousl\ shown to he maintained at tisl, timl Tc 12). thle1,, still appears t assess transport accurately In these tissues. Ihu1LS decreased amninoacid transport can be attributed to a loss of :absorptise sIllus cells, rather than to anleffect ott radiation onl the underix ing cellular transport mechaisms11.

Pres io us st id ieS of tlie efkct (if' radiation onl nutrient ahsorption b% rat intestinepostirradiation (3- -) r 'ported decreased intestinal absorption at 72 hi conisistent withthe results ot'the present stud\. In some eases (3-61. anl initial increase %%as observed

li \ \5()'t \D \) l( Rl IO1 SS RA0\IIN 429

At 1 1i thlat as not1 sCCil Inl the present sltldr RecedIC~ nutrient transport pOStirradia-thin1 \% as attII-hutcd to an inipatr nicnit o1 undrlsl 1%n, Cellular transport processes. I lti\k -C\ er . Inl suLpport Of-tilhe concIlusions' d ra\\ 1n Il thle present1 '4d ud reduced t ranisportoltoen coi ncided tIh sXcrcriopooa damalge to thle litest11,Inal ni tcosa ( 5. h~ ).

I kc\\ Ise. K\\ ock 'tI (I I 0bscrI\ ed I ha 't decreased \aI-deCpeitc nt in i n l O ac Id t ran's-port1- In)1\ IX mphIIo Itd ce ,-Ill' c i I aeIatCI\ I)rIcCcdCd decreased c-ClII\1,11 bIt\. WddIIot1,a Il p-p)oil o01 thle notion thlat deerescCd nntricntl tranSport does niot result tri- 11impaircdCclUlar trpr t pvOCW c s is on nd In More recent stud ics In M' hiCh tile ctlccts ot*radiationl onl tas ort anl bC ccarl\ dist Inl! Uished roln cell killing and eticsonprol Itcratloll. ( liccnian11 ,1 1! 5,) obscX cd nlochang: In) CIcuFcn Transport Inl cnterIo-cs \tes Isolated friontl rats 3 das s f'tcr6 (1\ g lucose tranisport %% as act nat en hanced Inlhese cell, Is hiiis latterl ctkct %%a, SU1!gCeted ito be COITT pc~nsal r Ssn bscucnIto d( loss

of' absorptit c snrhiceX area. Additionalls . Mioran cl (1/ ( IN) ohbscr' cd no chanic Inlbasal N\a-coupl1cd sug1ar transport ftlloMinc, I rradiTatilonl 01' cultured renal CpIthelialcells. I he.% obser~ ed. hokkcker. anl inabilit\ olirradiated cells, 1 5 TO l o p-reculatectc ie mII11I erot gi ncose t ransportcrs sk hcn 1oi \cn anl appropriatc st il in ni ns that ,\ as at-tribntd to an Clleet of radiation Onl gene expression.

In] addition to stndx ing nutrient absorption. tis stndx also examinesc, Intestinalsecretor\ proccsscs postirradiationl. Assuming (Lis Is gencrall\ acceptcd)I that theseprocesse .s reside Inclls I'of'thlC crrpt epitheli um (9-1it. csalniation of the response of'the tiSsne to a seCrela&'gi ' sueh aS thcoph llIine ,, Ill assess thle Fu nctional in teerit\In) terms 0ot transport)t of' this region. Thecophlline %\as obser\ ed to increase I, of'

control a elLsirradiated intestinal segmentsa carl\ tille Ot~O~c( 21)Ithle increase In I., elicited I\ theoph\ tine is related to a sti mulation of anionl secre-tory processes ( /3)- As is tine for osmotic thuid absorption reCsulting,- fromn alani netransport, this secretor\ process stimint aes thle scret ion o' tlid into thie intestinallumen1CI. Indeed. it is thle stimu Llation ofCCeLutzr secretor\ processes that is responsibleb'r fluid and electrol\ tec loss associated withl a large number of dliarrheal diseases (/19).

D)ecreased responlsi~eness of' lecal segments to seCretagogneCS \kas obser~ed 72 hpostirradiation. Ei: xaination of the morpholog\ of the intestinal mlucosa ait this timerev ealed blunting of' thle , illi and increased mitotic acti\ it\ in thle er\ pts. B\ 96 hpostirradiation. mitotic figures were observed even at the neck of the ervpts. hut amajor loss of CeltUlarit\ in thle crx Pt epithelium during this time was not apparent.Ihu1Ls. as described previously for the small intestine of other species (20-22). rabbitileum apparent)\ undergoes a prollelrative burst in anl attempt to repopulate the Inl-testinal mucosa. Evaluation of transport data w\ithi respect to the morphologr of thecrypt epithetiumi would suggestithat, in contrast to absorption. secretion is dimlinishiedin spite of' a maintenance of cellularits in the crypt epithetium. Since substantialblunting ol the villi does occur 72 h postirradiation. this result Could be interpretedas indicating that some villus cell population that is secretory is lost b\ tis time.Since amino acid transport (clearly a villus cell function) was not diminished at 72 libut was diminished at 96 h, this would require a prefe~rential dlecline ill secretoryprocesses and/or enhancement of absorptiv e proicesses In these cells. (i~en the ev I -dence suggesting that secretion is localized to the crypts (ef. (9-il)) and that increasedmitosis is observed at 72 h, a more attractive explanation for the data is that the

ilnabilit\ ol .tile tiss ut to respond to a secrctagoAIrc Is related to decreased diffren tia-Ilon ot thc cr\ pt cell population.

IcsC rcslts corn ~pIllcmct On rcarl icr obscr\ ations ( /2)f change,, in basal acti \cCcCctrol\I it ransplort postirrad ration i*\ sccrctor\ tresponisec as (ibstr\cd M i ichpeaked btts~ cci ll, and 24 h post irradiation and SnbhsC( nCntlII\ declined. lh ba"sot,til' ti11 inc conrsc \ as LTinc\plai ncd. It no\\ appcars r-casonal.1c to sUgtstC 1,hat1 &. torsccrction strrnnulatcd ill 1/n'K liohhlit thc basal ,ecrctor% rctsponc postirradra-tiori dcinCsIII dirt to popuL1ltion o1 thit rntcstirial cr\ pis %itli Itss, diritacecll,,

Ili riterpreting tilc rCsnlts of' tisl' sturdxs ji rpcct to tilec Ctlct of, r adiatiori oil11ICstirial1 I'Ictiori. t\%0 Irmportanit Clal Icatrtliirc considcratiori. I lit lirsit is that tile

clcitil cOniitionls ciiiplo\ced nIa'. not prtcisclk rcfcct tile Cii'\ iroilincInt ol thcIirradiated Initesti ne ill ill I Illis is a corISCIL nCnCC o 0Itlit ltl lni tiiciS rol rueasuri ng

aci\ctraril ,-In lar' Intest inal transport \01CI mi u nst bc donc Ill 111W nsliln a \s cI I-defi ned mcd a bathin ilit Iisnc. Ii nUS tilit t iNssnc IS not C' poCd to x arroUs substance,.normnallI' presen t inl tilit I~Ii cC to. bilIt and panicrcatic _n/\nmcs) or blood that nia,,onti buh~tt to alteration,, i in testi nal tranlsport postirrao-ation. lb1s concerni is tern-ptrCd. liur\\cs r. h\ tile fact that tilet tissues Used inl tisl' .rUind\ mert isolated hrorn tileanim11al imn11CdIlatcl\ bcforc transport 'was assessed and tiicrclorc c\poscd to thesc illin fachtors from tilit t imie of cxposnrt to t ti me ofrncasurnrnt. Prolonged effectsandlor daniage fromi snch agents \\ould still be obscrvcd Ii these stndics. and al-

thuh short-Ir \cd effects xsuld not be obsers cd. tlhe\ arc lcss likcl\ to be factors ithle response.

The second caveat is that the changes observed ma,. not reflect direct radiationdamage to thle intestinal inucosa. but secondlar,. teets of agents such as those dis-cussed abovc or other factors resulting from radiation exposure. Ont of these is thepossible influence of decreased f ood intake observed in irradiated animals which wasesaluated in this study by comparing the response of' fasted animals to alanine andtheophvllHine to that of' their matched controls in a separate series of' experiments.Diminished alanine and theophylline responseso Ifsegments from Casted animals weresignificant at 48 h but not at later times when significant effects were observed be-twNeen irradiated animals and controls. Although the magnitude of the differences inlthe response For irradiated animals was nearly twice that of fisted animals, a directcomparison of the data from these twAo groups did not reveal statistically significantdifferences between Casting and irradiation at 72 or 96 h. While this may be relatedto the degree of variance associated with the physiological response of the two groupsto these conditions, it prevents concluding unequivocally that decreased food intakeis not a lactor in the radiation response. However, considering that irradiated animalsare not totalli. fasted and have resumed eating bN 72 h postexposure. the direct com-parison of fasting and irradiated data may overestimate the role otfa~sting in the re-sponse of irradiated tissues. This taken together with previous studies of'starving rats(23, 24) indicating less severe morphological changes than those observed in thisstudy in irradiated rabbits makes it difficult to reconcile the results from irradiatedanimals as only secondary to decreased food intake.

In summary, the response of the intestinal mucosa to an actively transportedamino acid and a secretagogue was used to assess differential effects ofiradiation on

IIl\[ \BSORPII0\ \\l) SI (RI I k)N P(ISIIRR\l)I\IION 431

thuLs and erNIpt eplithelia. resp)ctil'sl. I hese rests Itaken together with the morpho-l ogical chtan ges obIse rsed su gest that secret or,. processes decl Ine at 72 h perhaps dueto ic reased prolIt erattIonl n the erspts at the expense ot'dI lerentiatIion. NutIrientI abh-sor1ptIOtl Vsa obsr' ehCI d ito declIine ILater at 96 hi a nd was related to the l oss o I mature

i1LkIS Cells V.' hiCh norniIall' ocup the intestinal 's thuLS.

V \MxNOI [tXONIFN I S

I hie altl ii tl hank, I I I cinan - \Akabh I r preixirat ion I Isse ISIC"tr l ight ni en )scoip\ I.\\ ilci\ nski I o

I L'hn[il .1assI tanC III transpori t It Aie, andI I )r J1 NoldI tor hielIpfulI diseussiotis concerning (fie morphoIitiig-1-1 ttdIes. Ibi IsI orIk isd as sppOrted b\ the \rmed [Firces Radtiihiolog\ Research Institute. D~efense

\ uclear \gcneo . uiider research ss ork unit NijiltI I'. thle \lcsss presented in this paper are those of thleautlhor: 1 n, eitdorscnent h\ the I )elIIC seNUclear \uenei\ has been gis en or shIould be intbcrred. Researchis as conducted ace rd ing to the pri nci pies enLITI nated in the "Guide for the ( are and I w (it I aborators\nInidI 0 prep~ared hi, the lnstiti oite il :horatirs .\nirnal Resources. National Research ( ounecil.

RI I l'\ IID: lebhrUar' 9. 19~)88: R1 \VlsI 1): September1 I .1988

R1 IERI. N( I S

). P. R iD. I \1I I 1 11 D\[ R . and J. 0. ANR( 1t.1 Hi M .1 tiiioiii/itii Rathion !.i it Acadenmic

Prs.Ness N ork. 19,I2 V .t \ it 1t-S\ii I it. I fleet ofl oniulnt! radiation oin gastirointestinal phi siologi . Inr ti/ai Rut/ia/i-

'I'e . j ( oiiklin and R, I. Walker. IdIs,). pp. 1 '5-1 51. \cadenti1C Press. Orlando. I-1 . 9X7.

1 tISt t I 1\ \\. Nbsiirpt ii n oft hegatro-i nestinai tract of the rat after N -irradiation. .1 in .1 l'/i-o201. IMi3-l]Mii1V'(l1o.

4 N~. 1). P! HHi'S. Iintestinal transpoirt and metabolism titlloss ing is hole-buds irradiation. Radial. Ret 29.

Bi . Ut ii i t m. ( . B. ( at H tRH and J. Di tROit. In s iso absor-ption ofcarbob' drate-. n rats with gastro-intestinal raiiat ion ss ndrome. hla Ruehii i/ 1hr /iI. Uthu/ 16. 97-96 II19771.

MI. NIf iti tttN_ K. [.s'sti R %. attd P Dt M vo . Changes in absor-ption ofglueose and proline follo"-Ing Irradiation to the esteriori/ed ileum. Radial RvN 74, I186- 1911(11975).

v N B. R. I iisi i\. C. 1. ( ftttss'st.x\, attd K. W-xt IKFR. Elkt of'abdominal irradiation on the kineticparameters ofiitetinial uptake of'gluteose. galactose. leueine. and gI's-leucine in the ratJ. l.au C/in.Vold 1012, 813-827 11993).

.C.I , ( it Ist 'st\ss. A. B. R. Ittimsit and K. W-Ms K[ R. The eflects of abdominal irradiation onitestitnal transport in the rat as assessed with isolated epithelia; cells. Raediat Res 101, 13 1-143

"S(I S( itI t I/. RW A. t Rt/F t . and HI. N. Nii~ I, \ Ion transport by mammalian small intestine.imo Ru, I/'li \/ 36, it-911(1974).

\1i NI. ii I it. In tracel ILiar mediators of secretion in the small intestine. In Ifeh/apasin ott IntieimtatNe a,w I 11. J. Binder. Ed. 1. pp. 8.1-9 1. Mlan R. tiss. Newr York, 1979.

IIR \ I R tni I I and S. (i. S( int t /. Models ofeleetrolyte absorption and secretion byI gastrointestinalepi tle lia. In lohi'rmwio na Ru t i i h oo, i/ Mn srou'lotahI/tioii v 111 (i~I R . K. Crane. Ed.),I .,It.) pp. 2Wt5-2 _'5. nis. Park Press. Baltimore. 1979.

/ P.1 (I,i I t R -5Sti II I. Iiitiing radtation alhcts actiise electroly te transport b\ rabbit ileum: Basal Naand ( I transport liiio /'/iih I 250 (Gatfloitu's Liter Ph/i bo/ 131. GS4t)-(i545 ( 19861.

\1 N I~t itt1. Ion transport in rabbit ilea[ mueti. If. Effects ofcyclic _3'.5-A N'P.. Jot. J1 Ilirvo 221.

14 .1 S. I R t R. Niirpholutg\ of the epithelium of the small intestine. In Ilandhou uuf l'Iieii/oii.' (C. V.

(itde. Ed.). Sect. 6. Vol. Ill. pp. 1125- 1175. Amn. Phystol. Soc., Washington. 196$.J .. M1 \ i,,iN. J. R. MNI s is;, a nd( A. [)1 1 Ni t.Vhe gast roi ntest inal t ract. I n Paiuith o it! l orrai/tuon (C. C.

Berdis. Id.). pp. 296- 344. Williams & Wilkins. Baltimore. 197 1.

43 N\1 I I1 , F R-S\I I It

I~ I\ I si' I if (In Ila 11 'i'~\ aigraphI I studis % t sugar fanId .a11i1n1 akdL hbs rtjo n hN

ex erlt I sac s ,It h in ste r I I est iii .1 (e/l'r 25. 111-',,) 9t , % ) a

deper lentI ailI no ai, d transport fin Furnait I H~l-tariFIuman B tRP\IF 1-8) mphoideclls

N '.I F)% s is arid \1 st\,\. F Ide III rakliatioii On the renIid in Ofslun-eendnIuL1,s1 transport II I I ( -PI 'K pithelal sellink-e. Po'sihi model for wire cspresiri kRaii lb

I' 1 -1 J ID R,~o \C1N fluid and LctiClsIte secretion. InII i'i iiiii Of iiin Nci'i iI Binder.t. pp 1, 11)11. Alan R. I iss. Net Nirk, 1979.

10 I I PIII Rand J lis t \I\. Rco e r% nI rcprodUtIc SC dctiSI\ \ ald th iIc nIILIaiIrc I st ruec tural fl leg-rit_ ot [ine intestinal epirteliumi ii the mouse after single dlose in hole-hnds btt-(ognmm ra\ espo-sure. FII /h', oi Raiwir i ,ti I 'Iit l'rolirililm dnflifrpi~,i ' 'ir' ia Simnp-,imon 11( i ',kdru'i' lr/a rl'ci~ 111 /,,j X/ ,reli pp 5 ()7_ F N.F l .

\ I CI)na. 1)6S2/ L C. 'I \t and A. H. ( siR1i . eclI-sum sal chatracteristics oF itiestimnal stem7 clls1 and cr~pts of Co-

irradiated mice. Riho Rt' 8(), 1'- 1() ( 1 1)t.22 k R IF I \si i. 1), F F Ift \\I\.( If R. R J. "Ll. [ N anid \ R. S \1 II sI. I hie repi I F nmall jintestinal

stem Lcell in the monluse to dmr-IS and irradiation treatment. lII (i'//l'r Ih'o 11 tIIIC 0,1\11 'Il

aal, 11,1,1/t F). R A pplcft ii J P. Surlter. an tilV .1. \at son. d.t.pp. 98-21 . Pit lniai Medical,Mlarshfield. I S.

2 N N. S r ft I. I R, I ORnt) Rid S. I S [RI It \\,ss. and S. J. (itN ,s 1E11ct of star~ation on the tissuecompOsition of the small intestine in the rat. Fm J1 /'/iionn/ 215, 75-77 11I909).

24I 1. S. [It fitsst and R. J. I I S i\. [flet (it semistariation onl the kinetics of actise and passise sagarabsorption across the smiall intestine in 'dso o1 Ph/ri s/ 252. 081 -70 ( 11974;).

ARMED FORtCE$ MAOOOLOGYAMARCH MOVITUTE

ScIENTIPIC EVOT

SR89-1 7From, TERRESTRIAL SPACE RADIATION AND ITS BIOLOGICAL EFFECTS

Edited D. VPrrciVa1 D. McCormz ('arles E. Swenberg,and Hor. tA,:,:ker

(Plenum Publisliing Corporation, 1988)

EFFECTS OF IRON PARTICLES ON BEHAVIOR AND BRAIN

FUNCTION: INITIAL STUDIES

Walter A. hunt, Bernard M. Rabin, James A. Joseph,

Thomas K. Dalton, Warren E. Murray, Jr., and Sherrie A. Stevens

Behavioral Sciences Department

Armed Forces iRadiobiology Research Institute

Bethesda, MD 20814-5145

INTRODUCTION

Successful operations in space depend in part on the performance

capabilities of astronauts and little is known about potential consequences of

exposure to ionizing radiation on behavior and the brain during manned

space flights. This possible threat has not been given much attention, since

all manned mission have been located in low equitorial orbit and radiation

there has not been considered hazardous. Future missions in space will

probably involve polar orbits, long-term space travel beyond the Earth, and

extended periods during which astronauts are operating outside their space

craft. Since exposure to radiation increases under these conditions because of

the absence of the Earth's normally protective transpolar magnetosphere,

astronauts may be placed at considerable additional risk. An understanding

of this risk may be vital to the survival and effective performance of future

missions in space. Therefore, it is desirable to understand the medical and

operational risks to personnel, including an assessment of possible behavioral

and neurobiological deficits.

Radiation hazards outside the protection of the Earth's magnetic shield

arise from solar flares and intragalactic cosmic rays. Intragalactic cosmic rays

are composed of protons, alpha particles, and particles with high charge and

537

....... ..

eniergy (IIZFE). suich as iron. Althoughi the hazards oif exposuire to cosmnic

rays are often (iiniiinizo'd t hey cani (etroy (Nistitig vels.. U<nlss their effects

catn soiiieiow b~e redhuice(i the( e'ffeCtS on the various organs, of tihe body,

rincludinig the brain, by their continuous bornziirdinezt by radiation during

long, space flighits couldI be disastrous. InI some instances, It has been

51 iggecsteil that the effects of cosinic rays on space travelers could rc~uilt in

ssiiiht onatologv resenbling those of Alzlieiiiier's or Parkinson's disease or

adlvanicing' age i ncluidinig significant cognitive and/or mnotor imipairmnenits.

Sich impihairmients 'oild( jeopa'rdize the successful comipletion oif a iniss IOTI

anid have ion-teriii consequences on the health of astronauts. l're~enitly. litt le

research has liven done to a(I~ress these issues.

Ciirisiuierablc a~lvariees have hJc('h over the last 20 years, in the st tidy of

behavior and their iieurobiobgliCal correintes- Specific p)aradligins are living

usftI to inv' t igatv thle effects, of ion iz inrg radliat ion on behavior (Mic kivy et

al. . 1988). as wvell as iivurochieiiical aiid neuirophiysiologic al enidpoints t hat

undi~erlie behavior undler study. By combining these analyses wvith vixery

sen-iitive behiavioral assessmients that can mieasure specific aspects of cogniitive

or motor performance following H ZE irradiation, more inforniat ion c an be

ob~tained concerning important biochemical and behavioral relationships that

will eventunally aid in predicting and controlling possible performiance deficits

occurring during manned space flight.

AFRRI RESEARCH IN SPACE BEHAVIORAL NEURORADIO BIOLOGY

G;eneral Considerations arid Design

In order to gain some insight into the possible behavioral and

neurological risks of irradiation in space, investigators at the AFRRJ initiated

a broad research effort using a variety of approaches. More specifically, the

general research program is designed to gain information on possible

alterations in behavior and the brain after exposure to heavy particles, such

as those that might be encountered during space travel. The objectives are

to describe andl characterize radiation-induced behavioral deficits, deterrine

their underlying causes, and develop approaches to minimize such deficits.

In May, 1987, the research group spent 3 weeks at the Lawrence

Berkeley Laboratory (LBL) in Berkeley, CA, assessing the effects of 000 MeV

538

5 6 Fe particles delivered by their Bevalac after doses of 50, 100, or 500 rads

on several behavioral and neurochemical endpoints (Table 1). These

measurements were performed at various times after irradiation (Table 2) in

an effort to obtain preliminary information on the potential hazards of space

travel outside the protective shield of the Earth's magnetosphere. The effects

of the three different doses of radiation were studied in rats at five different

times after irradiation (3 days to 6 months). Although all of the data have

not been fully analyzed (6-month groups), several important observations

were made. Presented here are the results of studies examining the induction

of a conditioned taste aversion by 5 6 Fe particles and the actions of these

particles on sodium channels in synaptosomal membranes. Motor responses of

animals exposed to 56Fe particles and the mechanisms in the brain

underlying them can be found in the paper of Joseph et al. (1988) in this

volume.

In the basic experimental design of these experiments, several

assumptions were made. First of all, in spite of the complexities of the

radiation environment in space, it is presumed that conditions in space can

be simulated on Earth under controlled conditions. Biological experiments in

space are very expensive, yield too little information when sensitive systems

have not yet been identified, and are generally impractical. Secondly, since

little is known about what space radiation might do to behavior and the

brain, effects found with other qualities of radiation are presumed likely to

be found after exposures to space radiation. This approach provides a

starting point for the design of appropriate experiments. Finally, since long-

term, low-level irradiations are impractical with the sources available, the

effect of a single dose over time is assumed to provide useful insights into

how space radiation might affect the behavior and the brain. This

assumption may be especially useful when studying the brain, since the brain

does not significantly repair itself after damage.

The irradiations were performed at the Lawrence Berkeley Laboratory

(LBL) with the remainder of the experiments completed at the AFRRI. Male

Sprague-Dawley rats were irradiated by 600 MeV 56 Fe particles delivered by

the LBL Bevalac. Iron particles were chosen because of their high LET and

the difficulty to shield against them. Doses of 50, 100, and 500 rads were

used to reflect the maximium exposures expected. Measurements were made

at, five time intervals after irradiation ranging from 3 days to 6 months in

order to look for acute and delayed effects.

539

TABLE 1

EXPERIMENTAL MEASURES

BEHAVIORAL ENDPOINTS

Taste Aversion Learning (Index of Nausea & Emesis)

Motor Tasks:

Inclined Screen (Muscle Tone & Stamina)

Wide Rod (Motor Coordination)

Narrow Rod (Motor Coordination)

Wire Suspension (Upper Body Strength)

NEUROCHEMICAL ENDPOINTS

Sodium Channels (Basic Neuronal Process)

Dopamine Release (Motor Function)

Catecholamines and Turnover (Brain Damage & Rate of

Information Transfer)

TABLE 2

GENERAL EXPERIMENTAL DESIGN

FOR MAY 1987 EXPERIMENTS

ANIMAL MODEL

Male Sprague-Dawley Rats

RADIATION SOURCE

600 MeV/nucleon Iron Particles (LBL Bevalac)

DOSES

50, 100, 500 Rads

DOSE RATE

100 Rads/min

TIMES AFTER IRRADIATION

3 Days, 8 Days, 14 Days, & 6 Months

540

General Methods

Five behavioral and three neurochemical endpoints were assessed in these

experiments (Table 1). These endpoints were chosen because of their

sensitivity to other qualities of radiation. The behavioral endpoints include

the conditioned taste aversion (CTA), an index of nausea and vomiting, and

several motor tasks, measures of muscle tone, stamina, coordination, and

upper body strength. The neurochemical endpoints include the movement of

sodium ions through channels, a basic neuronal process; dopamine release, a

regulator of motor activity; and catecholamine levels and turnover, a rough

estimate of brain damage and the rate of information transfer in the brain.

Male Sprague-Dawley Crl:CD(SD)BR rats (Charles River Breeding

Laboratories, Kingston, NY) weighing 200-300 g were used in these

experiments. Rats were quarantined on arrival and screened for evidence of

disease by serology and histopathology before being released from quarantine.

The rats were housed individually in polycarbonate isolator cages (Lab

Products, Maywood, NJ) on autoclaved hardwood contact bedding ('Beta

Chip' Northeastern Products Corp., Warrensburg, NY) and were provided

commercial rodent chow ('Wayne Rodent Blok' Continental Grain Co.,

Chicago IL) and acidified water (pH 2.5 using HCI) ad libitum. Animal

holding rooms were kept at 21 + 10 C with 50 + 10% relative humidity

on a 12-hr light:dark lighting cycle with no twilight.

The rats were irradiated with high-energy 56Fe particles (600 MeV) in

the Bevalac at the LBL. The animals were irradiated in well-ventilated

Plexiglas holders with one of three doses, including 50, 100, and 500 rads,

at a dose-rate of 40-140 rad/min. Dosimetric support was provided by

personnel at the Bevalac. Animals irradiated with other radiation sources

were exposed to a single dose of 50, 100, or 500 rads of gamma photons

from a 60Co source at a rate of 40 rads/min or high-energy electrons (18.6

MeV) from a linear accelerator. Radiation dosimetry was performed using

paired 50-ml ion chambers. Delivered dose was expressed as a ratio of the

dose measured in a tissue-equivalent plastic phantom enclosed in a

restraining tube, to that measured free in air.

General Observations after 56Fe Irradiation

Although not a specific part of the experimental design, any unusual

541

reactions by the animals were noted. Based on subjective observations, the

animals appeared normal after irradiation with 56Fe particles. However, after

exposure to 500 rads, several changes were observed. The exposed rats

progressively lost weight, totaling about 20% of body weight over a 14-day

period. In addition, they experienced a reduction in muscle tone, a hind

limb tremor, arid hypothermia (animals cool to the touch) 3 days after

irradiation, effects that had disappeared by 8 days after irradiation.

CONDITIONED TASTE AVERSION LEARNING AFTER IRRADIATION

Characteristics of the Conditioned Taste Aversion

Animals have developed over the course of evolution mechanisms to help

prevent accidental poisoning, the best-known one being the emetic response.

Emesis can occur as a result of consuming presumably tainted food that iL

then expelled from the stomach. In addition to emesis, animals are also

capable of avoiding potentially toxic substances after a single ingestion of

quantities less toxic than those required to induce vomiting. This is done

through a process called the conditioned taste aversion (CTA). A CTA

develops when the animal associates the taste of novel tasting food with a

physiological response, possibly illness, and then subsequently avoids further

ingestion of that food. In a laboratory setting, a CTA is typically induced

by pairing a normally preferred but novel tasting fluid with exposure to a

toxin. The animal will then avoid drinking the fluid when presented again.

The conditioned taste aversion (CTA) paradigm in the rat can be used

as a model system to study the mechanisms by which exposure to non-lethal

levels of ionizing radiation can produce changes in the behavior of an

organism (Rabin and Hunt, 1986). Because the functional effects of emesis

and taste aversion learning are similar, in the sense that they limit the

iniake and/or absorption of toxic substances, a number of investigators have

argued that the CTA paradigm represents a model system for the study of

radiation-induced nausea and emesis (Garcia et al., 1985; Rabin and Hunt,

1986). Therefore, the CTA provides an index of the probability that nausea

and emesis will occur.

542

The CTA paradigm offers a number of advantages over emr"t iz m-dls.

1he paradigm can be used with rats, inexpensive and easily used animals.

They are small enough that more uniform fields of radiation can be obtained

with particle accelerators than with larger animals. Because a CTA can beeasily induced in a relative large number of animals, a great deal of

information can be accumulated quickly as well as the characterization of

any responses. Since the mechanisms underlying the CTA and emesis appear

to be similar, this approach will allow for the formulation of more specific

hypotheses that could be applied eventually to emesis models.

The CTA induced by ionizing radiation has been extensively studied and

a clearer idea of how it develops has been emerging. The most important

discovery is the involvement of a specific nucleus in the brain stem, the area

postrema. The area postrema has been demon rated to play an critical role

in the development of CTAs induced by a broad range of '-irelated toxins.

This part of the brain is sufficiently important that if the area postrema is

lesioned, the development of a CTA is blocked. These toxins include not

only ionizing radiation (Ossenkopp, 1983; Rabin et al., 1983), but also

lithium chloride (Ritter et al., 1980; Rabin et al., 1983), copper sulfate

(Rabin et al., 1985), aluminum chloride (Rabin and Hunt, unpublished

observation), paraquat (Dey et al., 1987), angiotensin 11 (Rabin et al., 1986),

amphetamine (Rabin et al., 1987), WR-2721 (Rabin et al., 1986), and

cisplatin (Rabin and Hunt, unpublished observation). In addition, other

evidence indicates that the area postrema is also required for the

development of emesis (Wang et al. 1958; Brizzee, 1970; Harding et al.,

1985; Rabin et al., 1986). Not all toxic drugs induce CTAs through the areapostrema. For example, ethanol- and morphin2-induced CTAs are not blocked

by lesions of the area postrema (Hunt et al., 1987; Rabin and Hunt,

unpublished observation).

Since many unrelated toxins induce CTAs through the area postrema, ;t

has been suggested that a common mechanism may underlie all these effects

(Rabin and Hunt, 1986). Also, since toxins are generally foreign substances,specific receptors for each possible toxin are not likely to have evolved.

Consequently, an intermediary mechanism in the induction of CTAs and

emesis involving one or more secondary mediators has been postulated (Hunt

et al., 1965; Rabin and Hunt, 1986). If these mediators interact with

receptors in the area postrema, they may then activate the neural circuits

that evoke CTAs.

543

Induction of a CTA after Exposure to 56Fe Particles

Research was initiated to determine whether high-energy iron particles

could induce a CTA similar to other qualities of ionizing radiation, such as

gamma photons. The first experiments were designed to find the doses of56Fe particles that would induce a CTA and compare the sensitivity of the

animals to those irradiated with gamma photons or high-energy electrons.

Conditioned taste aversions were produced by first placing the rats on a

23.5-hr water deprivation schedule for 5 days during which water was

available for only 30 min daily during the early light phase of the diurnal

cycle. On the conditioning day ((lay 6), the rats were presented with a

solution of 10'. sucrose, after which the intake of the fluid was recorded.

Imediately following the drinking period, rats were irradiated with the

doses stated above. On the following day (test (lay), 10%6 sucrose was

presented again and the consumption during a 30-main period was recorded.

A CTA existed when the amount of fluid consumed on the test day was

significantly less than that consumed on the conditioning day.

25

20T I COND

is / TESTSucrose

Intake (ml)10

5

control 50 100 500

Dose (rad)

Fig. 1. Conditioned taste aversions after different doses of 56 Fe ions.

Sucrose intake was significantly reduced after all doses

studied. The maximum effect of the radiation is presumed

to be < 50 rads.

544

140 -

120 2 5 6 Fe

100 . 6UCo

% Conditioning o e-Day Sucrose 80

Intake60

40

20 -

0 [,sham 50 100 500

Dose (rad)

Fig. 2. Conditioned taste aversions after different (loses of 56Fe ions.

gamma photons ( 60Co), and high-energy electrons (LINAC).56Fe ions were at least 10 times more effective in inducing

a CTA than do gamma photons or electrons.

Exposure to all doses of high-energy 56Fe particLc. tested resulted in

significant dose-dependent reductions in sucrose intake, indicating the

development of a CTA. In fact, maximum reductions were found after all 3

doses of radiation (Fig. 1). For comparison, when rats were exposed to these

same doses of gamma photons or high-energy electrons, dose-dependent

decreases in sucrose intake were observed, indicating the development of a

CTA (Fig. 2). However, 56Fe particles were considerably more effective than

the other two qualities of radiation. A maximal CTA was observed only

after exposure to 500 rads of photons or electrons. A further characterization

of the dose-response effects of 56Fe particles compared to other qualities of

radiation is currently in progress.

CEREBRAL SODIUM CHANNELS AFTER IRRADIATION

Characteristics of Sodium Channels in the Brain

The generation and propagation of electrical impulses or action potentials

in neurons is initiated by the rapid inward flow of sodium ions across the

545

neuronal plas-iia iniinbra xe (Hodgkin arid Huxley, 1952). In the resting state

a neuron iii int a is a ('Stinig iiieiiiratie potential that results from thle

Unequal dis trib 01 u~n of soiimni pot assiuimf arid chloride i01ns across the

memibrane ( hocs!er. 198 ia~l). Wh len neurons are electric ally excited, s0(1 un

ions flow inlward onl their concentration gradient until the nienibrane

potential is revers ed (Koester, 1981c) . The movemient of p~otassiumn ions out

of the n('uron proceeds until the neuron has repolarized anid the neuronl is

againl in the resti ig state.

Sodliumi ions enter the neuron t hrough pores in thle menibrane called

channels. These channels are spec ific to sodiumi and( traverse the neuironalI

lplasmia mnemibrane. They are glycoproteins containing multiple suhiiits

Catterall . 19,-2) anid have an absolute requ iremlenit for lip ids for normal

functioning- (Taikin et al., 1984). At least thlree functional sites xithIiin

sodiutm channels have been identified based onl the actions, of spiecific

neirotoxins (Catterall. 1980). Site 1, located on the externial surface of thre

lieuironal iniembirane. binds tet rodotoxin andl saxitoxin, dIrugs tliat block thle

gueneration of action potentials. Site 11, located in the lipid core of the

mnemrbrane. binds batrac hotoxin anid veratridine, lipid-soluble (I r igs thIiat

activate sodium chiatinels. And site III, located on the niemibrane surrfacc but

with projct ions down to site JI hbinlds scorpion and] sea anejilone toxinis t hat

enhance the act ions of toxins, oil site II butt have no intrinsic activity of

their own. Neuirochiemiically. tilie functioning of thle sodi um channel can be

studied withI a syniaptosollral (pinchi-off nerve etidings) preparation (Kruleger

anid fllatstein. 1980; Tankin antI Cat terall, 1981). The rate of tiptake of

2Na can be tme(asuredl after exposure to thle netirotoxins batrachlotoxiii or

veratrirliie, thereby assessing what would occur unider normalI phiys iological

conid itions.

Sodiuim Channel Fuinct ion after Exposure to 56Fe Particles

The rate of 2 2 N a upt ake was dIeterniined in synalptosoiiies from thle

cerebral cortex as (let ailed previobus ly (Mu tll in et al., 19 86). .A c rudiIe

tiitoclhotidrial preparat ion contain ing- synlaptosorties was ptreptared from a

ctortical homnogenate. The fitial pellet was resuispendled in ice-cold inc ubat ion

btiffer (8-10 mI/brainl) containing 5.4 mM.N KCI, 0.8 niM Mg-SO 4 ' 5.5 mMN

glcose, 130) nM choline chloride, arid 50) mM1 N-2-hiydroxyethiyl-pipe(raziiic,-

N'-2-ethianesulfonic acid (HIEPES), with the p11 adjusted to 7.4 withI Tris

base. The uptake of 22Na was meastured as follows. Aliquiots (50 pl) of the

sytiaptosonial suspension were incumbated for 2 min at 360 C. The ricorotoxin

546

veratri~lirie %%a;s then add~ed1, and tile incubation was continuredi for an

additionial 10 mill. The samrples were then diluted with 300 ul of uptake

soltion containing 5A niM Kt 1, 0.8 ruM~ NMg;O 4 ' 5.5 in~l glucose, 128 miM

chioline chloride. 5 mM~ ouabatin, 2 iuM NaCj, 1.3 #C 2 2 Ni 100) #%1

veratridirie. arid 51) mM IIEP§M (phI adjusted to 7.4 with Tris). After a

5-tec iricubitron, uptake %vas tcrnri.mate(1 by adclimig Imli of ice-cold wash

soluitioin conitainling 163 n\t chioliii( chloride, 0.8 ruN! NgS() 1.7 ruN

(a('l) I trg/rid of bvine scrumri albiriiii arid 5 A! HEIUIEs, (ii) adjuste-d

to 7.A with Tris . Irel riixtiire was rapidls filtered runder vacuumni thruigh a

celrldo,e filter wvith 0..ta-jyii port-. rirol I lic fll(rs were washed~ t wice withI 3

mil of' washi sollitiori. lRadjoactivitv wa detcriiied hV liquiid sciiitillationi

SI~t ropiiitutietry. Thel( da~t a were (xpress(d ats spiecific iuptake ulterinir, I by

stilhtractirig inonispif~ic ript ik (santiples couitiiiiti'' I LAN! tet roilttxin) fronm

total ut a ke.

1111 1 0 r ij

*500 iid

)r o tI i;n 10k

3 8 14 G Monith.(jays

Fig. 3. 22Nit uptake after different dloses of 56 Fe particles anid

iiifTerent intervalIs after irradliation. No Sign ifie ant differences

were oibservedl tinder anly of the experimental COTidit ions

sturid.

Pats were irradhiatedl with 56Fe particles in the mrannier dlescribedl above.

Mteasiiremients oif ve ratriliiei(-stimiiilite il soimi uptake in synaptoisoics (lidl

iiot reveal any conisisteiit anid statistic ally sign ificanit chianges after exposuire

(if aniy of the four (loses of radliatiTmi nor at any of the three initervals ' ft er

irradiation (Fig. 3).

547

GENERAL DISCUSSION OF RESULTS

The experiments completed to date demonstrate that 56Fe particles can

induce a (TA that may represent a state of illness, possibly nausea. These

results along with those presented by Joseph et al. (1988) suggest that the

brhavioral effects of exposure to 56Fe particles may be at least 10 times

greater thali those observed following expcsure to garnma photons or high-

energy electrons. They also may reflect a greater chance for the occurrence

of nausea and einesis in astronauts exposed to a space radiation environment

during longer tern missions.

The successful completion of missions in space dep~ends inl part on

behavioral and neural integrity of the astronauts. Given the potential

si'nificance of the data presented. it is important to seriously pursue an

active prograli of research into the possible behavioral and neural detficits

that might occur in space as a result of exposure to radiation. By

comiining sensitive Ibehavioral assessments that can measure cognitive or

motor performance with ricurochemnical analyses following HZE irradiation

(e.g. Joseph et al., 1988), more information can be obtained concerning

important behavioral and biological relationships that will aid in predicting

and controlling possible performance deficits during manned space flight.

ACKNOWLED(;EMENTS

The authors wish to acknowledge the assistance of Drs. E. John

Ainsworth, Patricia Durbin, and Bernhard Ludewigt and the staff at the

Lawrence Berkeley Laboratory without whose help these studies could not

have been undertaken. This research was supported by the Armed Forces

Radiobiology Research Institute, Defense Nuclear Agency, under work units

B-1098. B4123. and B4157. Views presented in this paper are those of the

authors; no endorsement by the Defense Nuclear Agency has been given or

should be inferred. Research was conducted according to the principles

enunciated in the 'Guide for the Care and Use of Laboratory Animal

Resources, National Research Council.'

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the area postrerna in radiation-induced taste aversion learning and emesis

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551

AmO POaCUS "AOOILOGY

ICBWTIMg RhDOfT

SR89-18From! TERRESTRIAL SPACE RADIATION AND ITS BIOLCGICAL EFFECTS

Edited by Percival D. McCormack, Charles E. Swenberg,and HorsL i;,cker

(Plenum Publishing Corporation, 1988)

CORRELATIVE MOTOR BEHAVIORAL AND STRIATAL

DOPAMINERGIC ALTERATIONS INDUCED BY 5 6 Fe RADIATION

J. A. Joseph, W. A. Hunt, B. M. Rabin, and T. K. Dalton

Department of Behavioral Sciences

Arined Forces Radiobiology Research Institute

Bethesda, MD 20814-5145

INTRODUCTION

In recent years there has been increased interest in the study of

mammalian organismic responses to heavy charged particles (HZE's). This

interest has been generated from a variety of areas including space

radiobiology (Grahn, 1973) and radiotherapy (Castro et al., 1985). While a

great deal of information has been generated that is concerned with the

mechanisms of HZE damage and repair (Curtis, 1986; Tobias, 1985) or the

early and late effects of HZE damage (Ainsworth, in press), there have been

few attempts to investigate the effects of these particles on any motor

behavioral parameters. The potential for HZE's to alter behavioral

performance becomes increasingly important when one considers that future

space travelers, especially those who will be on long space voyages, or those

who may be carrying out tasks outside the shelter of a space vehicle, may

be exposed to HZE's that can create microscopic lesions in virtually all

organs of the body (see Todd, 1983 for review). The questions as to whether

or not such exposure results in performance deficits and the possible

relationship of these deficits to the structural specificity of damage to the

brain have not been addressed very extensively. There are indications,

however, from at least one experiment (Philpott et al., 1985) that mice

given brief exposure to low doses of 40Ar particles showed time-dependent

reductions in performance on a wire suspension task. Moreover, if one can

extrapolate from studies in which organisms have been exposed to other

553

sources of radiation and behavioral performance examined, it might be

postulated that decrements in such indices as motor behavior are a distinct

possibility.

Several previous studies have indicated declines in motor performance of

iir rtiateci anituals plak-L d in ta-,s for tilc tc urgj; i., in Is required to

display physical strength, endurance, and coordination. For example, studies

in irradiated lower animals assessing performance on psychomotor tasks have

shown dose-dependent deficits (e.g.. equilibrium platform, Barnes, 1966, 196S:

accelerod, Bogo, 1984; activity wheel, Franz, 1985). These studies gcnerally

have utilized a variety of radiation sources ranging from mixed meutron-7

(e.g., Franz, 1985) to high energy electrons (Bogo, 1984). The prccise central

locus of these deficits is still in question. There is, however, a great deal of

evidence from non-irradiated organisms to suggest that the nigrostriatal

system may be very important in mediating these types of motor behaviors.

It is becoming increasingly clear that the telencephalic end termiintis of

this system, i.e., the striatumn, is one of the basic central processing areas

involved in the mediation of motor behavior. This structure appears to

control a wide variety of motor responses ranging from the simple (balance

and coordination, see Steg arid .Iohnels, 1979 for review) to the complex

(e.g., the ordering and sequencing of intricate behavioral patterns directed by

cxtcroceptive stimuli, Cools et al., 1984; Vrijmoed-de Vries & Cools, 1986).

Since it can be postulated that control of such complicated patterns of

behavior depends upon intrastriatal coordination of a host of

neurotransmitters and neuromnodulators, it would follow that if any of these

interactions are altered as a result of heavy particle bomibardment in a space

environment, decrements in motor function may result.

Of the many intrastriatal neurotransinitter systems that may be

involved in mediating these types of behaviors, two of the more important

are the dopamine (DA) and acetylcholine (ACh) systems. It is believed that

these neurochemicals exert part of their control through reciprocal inhibition.

Any radiation-induced morphological or pharmacological alterations in their

function may be translated into decreased reciprocal inhibitory control (RIC).

554

One area where this control becomes especially salient is concerned with the

cholinergic regulation of striatal DA autoreceptors. Striatal DA autoreceptors

are inhibitory, control DA release (Cebeddu and Hoffmann, 1982; 1983;

Cohen and Berkowitz, 1976; Dubocovich and Weiner, 1981; Kelly, 1981;

Plotsky et al., 1977; Westfall, et al., 1976) and may be of the D2 subtype

I,.: and Zahniser, 1986). If the)- are stin'ilatcd v;- DA aguits (e.g.,

apomorphine) or inhibited with DA antagonists (e.g., haloperidol), K+-evoked

release of DA will be respectively inhibited or enhanced. These receptors are

in turn controlled by a group of inhibitory muscarinic heteroreceptors

(Lehmann and Langer, 1982; Raiteri et al., 1982; 1984). Muscarinic agonists

can activate the heteroreceptors, which inhibit the DA autoreceptors, and

potentiate the K+-evoked release of DA from the striatum (Lehmann and

Langer, 1982; Plotsky et al., 1977; Raiteri et al., 1982).

In the present study, it became of interest to determine if there could

be a radiation-induced reduction of autoreceptor function that could be

related to the deficits in motor behavior that have been observed in

irradiated animals. The present experiment was directed toward making these

observations by examining motor behavioral performance in rats exposed to

HZE's and relating the degree of alteration of such performance to any

observed deficits in muscarinic enhancement of the K+-evoked release of DA

from superfused striatal slices.

METHODS

Animals

Male Sprague-Dawley Crl:CD(SD)BR rats (N = 180, Charles River

Breeding Laboratories, Kingston, NY) weighing 200-300 g were used in these

experiments. The rats were housed at the vivarium at the Lawrence Berkeley

Laboratories, Berkeley, CA. The rats were maintained in polycarbonate cages

which contained autoclaved hardwood contact bedding ("Beta Chip"

Northeastern Products Corp., Warrensburg, NY). They were given food and

water ad libitum. The animal holding rooms were kept at 21 + 10 C with

50 + 10% humidity.

555

Procedure

A. Radiation

The animals were divided into two groups (irradiated and control). The

irr 4itcd g-',p wa? furtber ,,ibdivided irto three groaps Ind ;rradiate,

with one of three doses (0.5 Gy, 1.0 Gy, and 5.0 Gy) of 600 MeV 5 6 Fe

delivered at maximum dose rate by the Bevalac at the Lawrence Berkeley

Laboratory. Control animals were given sham exposure by being placed into

a well-ventilated plastic restrainer for an equivalent length of time as that

for the irradiated animals. Only one dose was administered on a particular

day; so, the three doses were administered over three days. Behavioral and

biochemical changes were then examined at 3, 8, and 14 days post-exposure

or sham exposure in subsets of these animals (N = 10 control and 10

experimental animals at each time). Because of limitations imposed by other

in vitro biochemical tests (not discussed here), one-half of each group was

tested in the a.m. and the other half in the p.m.

B. Behavior

The animals were tested on a battery of four motor tests:

1-2. Rod walking -- The ability of the rats to balance on stationary

horizontal rods of two diameters: 12 mm (narrow rod) and wide 25

mm (wide rod) suspended 6.0 cm above a table was assessed. Each rat

was given one trial and could achieve a maximum score of 60 sec

(wide rod) or 120 sec (narrow rod). Animals were placed in the center

of the rod and parallel to it. The time in seconds that the animal

remained on the bar was recorded with a stopwatch. When the animal

fell off the trial ended.

3. Wire suspension -- The prehensile reflex refers to an animal's ability to

grasp a horizontal wire with its forepaws and to remain suspended. It

is presumed to be a measure of muscle strength. In this test,

individual rats were raised to an elevated hanging wire 8 cm above the

table top and given one trial each, with total hanging time (in sec)

recorded.

4. Inclined screen -- This test is purported to measure muscle tone and

stamina. Each subject was placed in one of 5 separate compartments of

a wire mesh screen inclined at a 600 angle. The screen was suspended

60 cm above the table surface and the maximum time allowed on the

screen was 1800 sec.

55A

C. Muscarinic Control of Dopamine Release

Following the behavioral tests the animals were killed by decapitation,

the brains quickly removed, and the striata dissected on ice. Slices (300 m/4)

were prepared using a Mcllwain tissue chopper. Striatal slices from each

animal were placed into small glass vials containing a modified Krebs-Ringer

basal release medium that had been bubbled for 30 minutes with 95%

02/5% CO 2 and contained (in mM) NaHCO 3 21, glucose 3.4, NaH 2 PO 4 1.3,

EGTA 1, MgCI 2 0.93, NaCl 127, and KC1 2.5 (pH 7.4). The slices were

washed twice in tb-is medium and aliquots placed into two chambers of a

superfusion apparatus contain Ing 16 parallel chambers. Since the apparatus

only contained 16 chambers, only striata from 8 animals could be

accommodated. Therefore, striata were examined from a!! of "l cxperimental

animals (5) and three of the five control animals (randomly selected) for

each run. The tissue and buffer media were maintained at 370 C throughout

the course of the experiment. Following placement into the superfusion

chambers, the tissue was allowed to equilibrate for 30 minutes. It was

continuously perfused with th: oxygenated basal release medium at the rate

of 124 p/min. After the equilibration period, a 5 min baseline fraction was

collected on ice and the medium was then switched to one containing (in

m.M) KCl 30, CaCI2 1.26 and NaC] 57 as well as the other components

described above (pH 7.4) in the presence or absence of 500 #M

oxotremorine. Following the switch, 5 min fractions continued to be collected

on ice for 30 minutes.

The fractions were collected into tubes containing 0.3 ml of cold 0.4 N

perchloric acid, 0.05% sodium metabisulfite, and 0.1% EDTA. These samples

were then stored at -80 ° C until later analysis for DA via high performance

liquid chromatography (HPLC) coupled to electrochemical detection.

The HPLC consisted of a Varian model 5000 ternary chromatograph, a

Varian 401 data system, a Varian model 8055 autosampler, and a Valco air

actuated injector with a 50 jul loop. The effluent was monitored with a

bioanalytical systems LC-4B amperometric detector using a glassy carbon

electrode. The detector potential was set at 0.72 V as a Ag/AgCI 2 reference

electrode with a sensitivity of 10 nA/V. The mobile phase consisted of a

filtered, degassed 100 mM KH 2 PO4 buffer containing 3 mM 1-heptanesulfonic

acid, 100 #M EDTA, and 8% (V/V) acetonitrile (pH 3.6). The components

were cluted off a Waters 10 pm particle, ufBondapak C 1 8 reverse-phase

557

column (30 x 0.39 cm; flow rate = 1 ml/min) maintained at 300 C. Results

were calculated relative to known previous standards that were analyzed on

the HPLC under similar conditions. Data were expressed as pmoles/mg

protein as analyzed by the Lowry procedure (1951).

Data Analysis

Data from the behavioral experiments were analyzed by using 4 (0, 0.5,

1.0, 5.0 Gy) by 3 (day) analyses of variance. Data from the perfusion

experiments were analyzed by first computing difference scores by subtracting

the pmoles of DA released to 0 pM oxotremorine from that released to 500

#sM for each fraction and for each "striatal-channel" pair. These difference

scores were then analyzed by 2 (control vs radiation) by 3 (day) by 7

(fractions) repeated measures (on the last factor) analyses of variance (one

analysis for each radiation dose level). In addition, correlational analyses

were carried out by correlating the peak DA release difference with the

number of seconds that a particular animal remained suspended on the wire.

The wire suspension task was used, since preliminary data analysis inlicated

that there were no significant radiation-induced deficits on the other

behavioua! tasks.

RESULTS

Behavior

The results of the wire suspension task are illustrated in Fig. 1. As

can be seen from this figure, exposure to 56Fe particles induced deficits in

this task, and irradiated animals remained suspended on the wire for a

shorter time than controls (F (3/142) = 26.09 p < 0.001). There were,

however, some differences in performance among the various radiation-treated

groups especially at three days post-irradiation, where the animals that

received 5 Gy did not show any deficits in performance on this task

(Duncan's post test comparisons, 5 Gy vs control p> 0.05, 5 Gy vs 1 Gy

p< 0.01, 5 Gy vs 0.5 Gy p< 0.005 df=11). At later time points (8 and 14

days), there were no differences among the radiation-treated groups (all

comparisons p> 0.05), and all differed from controls (Fig. 1).

558

M ControlM05 GyE3 10 Gym50 Gy

30Wire

SuspensionTime 20

10

03 8 14

Days Postirradiation

Fig. 1. Total time (in sec) that animals given ohie of three

radiation doses or sham irradiated could remain on a taut,

plastic coated wire with their forepaws as a function of

days post-irradiation

Results from the other behavioral tests indicated that performance of

the irradiated groups did not differ from the control group on the wide rod

or the inclined screen. However, there was a tendency for a greater number

of the irradiated animals to fall off the narrow rod before the 120 sec

maximum, especially at the later post-irradiation tiaes (e.g., 5 Gy vs control

at 14 days post-irradiation, 6/10 and 6/6, respectively).

DA Release

Analysis of the K+-evoked release of DA at the 0 juM oxotremorine

concentration indicated that striata from the irradiated groups showed

greater release than controls (0.5 Gy, F(12,468) = 2.58 p< 0.0025; 1.0 Gy,

F(12,462) = 4.37 p< 0.0001; 5.0 Gy, F(12,468) = 2.71 p< 0.01) and

subsequent post hoc t-tests indicated that this difference was especially

salient at 3 days post-irradiation. At later time points it was variable (Fig.

2). However, even though DA release to 30 mM KCI without oxotremorine

was higher in irradiated animals (Fig. 3), analysis of oxotremorine

enhancement of K+-evoked release of DA indicated that such enhancement

was reduced in the irradiated groups (overall radiation vs control by

fraction, 0.5 Gy F(6,468) = 31.36 p< 0.0001; 1 Gy F(6,462) = 17.54 p<

0.0001; 5 Gy F(6,468) = 9.72 p< 0.0001).

55t

100 . CONTROL 0.5 Gy Days

80. Postirradiation

60- x- 340 8

pmoles 4DA/mg 20 -ji,

PROTEIN 0 J""

100 1.0 Gy 5.0 Gy

80- x60-40"

20

0 12 46-2 4 61 2 3 4 5 6 7 1 2 3 4 5 6 7

5 MIN FRACTIONS

Fig. 2. Peak release of dopamine (DA) from superfused striatal

tissue slices following stimulation by 30 mM KCL. Striata

were obtained from Sprague-Dawley rats given one of three

doses of 56Fe or sham- irradiated and killed by

decapitation at 3, 8, or 14 days post irradiation.

560

120mConitrol

100 10 5Gy

i50 Gy80

prides ... ..DA,,mg 60

PROTEIN

40

20

03 8 14

Days Post~rradiitoon

Fig. 3. DA release to 30 miM KCI superfused onto striatal slices

as a function of days post- irradiation or sham- irrad iation.

(See Methods).

561

There was some indication, however, that the striata obtained from the

group treated with 5 Gy initially (3 days) showed no reduction of

enhancement of K -evoked release of DA. These deficits did not appear

until the 8- and 14-day post-irradiation time points (radiation vs control by

fraction by day F(12,468) = 5.70 p< 0.0001). In contrast to this group,

attenuation appeared as early as 3 days in the other two radiation-treated

groups, and response differences as a function of days were not observed

(F's < 1, Fig. 2).

Interestingly, these response characterizations parallel the deficits

observed with respect to wire >uipecnsion. This is more clearly seen by

comparing Fig. 4 which shows the peak enhancement of K+-evoked release

of striatal DA with Fig. 1. It can be seen that the response profiles from

both the biochemical and behavioral parameters are very similar. For

example, the L Gy group showed neither behavioral deficits nor DA response

reduction at 3 days post-irradiation, while at 8 and 14 days, deficits were

observed in both response parameters. Subsequent correlational analysis

indicated significant correlations between the time on the wire suspension

task and degree of oxotremorine enhancement of K+-evoked release of DA

from the striatal lices (Fig. 5, 6, and 7) from the irradiated aninmals.

120M Control

100 ' 05 GyE10 Gyi50 Gy

80pmolesDA k.'mg 60

PROTEIN40

20

03 8 14

Daiys Po. tir r (aFitiorn

Fig. 4. Peak differences in DA release between 0 u*l and 500 'UI

oxotremorine superfused onto striatal slices in the presence

of 30 mM KCI is a function of (lays post-irradiation or

sham-irradiation.

562

DISCUSSION

The present results elucidate one possible mechanism by which motor

deficits may occur following irradiation. It appears that there is an

attenuation of muscarinic heteroreceptor responsivity in the striatum that is

maximized in all groups within 8 days after exposure to 56Fe. This change

is significantly correlated with at least one of the motor behaviors which

assesses upper body muscle strength (i.e., wire suspension). There were also

0120100 o0o

.o 0. . .80 0

0 0D A n i g 6 0 -- ' ' o

PROTEIN 60 a "

0. 000

2 .20 c) e - 07.pO 0 1

s- Radiation (r 071. p 0001)0o ... Control (r 034, p 0 005)

0 I 1LL I L... ILJ0 0 20 30 40 50

Wire Suspension Time (sec)

Fig. 5. Scatter plot of peak differences in DA release between 0

/sin and 500 #uM oxotremorine superfused onto striatal

slices in the presence of 30 mM KCI for each animal as a

function of its wire suspension time at the 0.5 Gy level.

deficits in the irradiated animal's ability to remain on the narrow rod which

were greater as a function of the post-irradiation day on which they were

tes~ed. While the mechanism of these deficits is not clear, it has been

hypothesized that these changes may be brought about by the destructive

capabilities of free radicals generated by the irradiation (See below). If this

is the case, then there should be other instances, perha ps in nonirradiated

animals, in , which damage from free radicals would produce similar kinds of

effects.

563

220200 -0180160140120

pmoles 120 - 0....

OA/mg 100 0 0

PROTEIN 80 - o. . 0

4020 600 O,

0 - Radiation (r = 066. p 4 0001)-20 o .... Control (r 044. p 4 005)-40-60 t

0 10 20 30 40 50Wire Suspension Time (sec)

Fig. 6. Peak differences in DA release for each animal as a

function of its wire suspension time at the 1.0 Gy level.

160 - •0

140 -

0-Rditin r 01-p" 001

1200. C

100pmoles 10 0 30 ., 4 5 6DA/mg 80- .1° 0

PROTEIN 06 0 -0• 0

40- .0_0 .020 Radiation (r

= 051, p 4 0001)

20 0• 0 ------. Control (r = 0 40. P 0 0 05)

- 1 0 '1 . l 1 1 1 1 ' I 1 1 1 1 1 1 I_

0 10 20 30 40 50 60 70

Wire Suspension Time (sec)

Fig 7. Peak differences in DA release for each animal as a

function of its wire suspension time at the 5.0 Gy level.

564

One group of organisms where damage from free radicals seems to have

taken its toll on central nervous function is made up by those that are

senescent. It is believed that life-span effects of free radicals produced during

the course of normal metabolism are believed to be responsible for the

alterations in membrane structure and function seen in senescence (Schroeder,

1984). In the CNS, the transbilayer fluidity differences induced by

peroxidation may be intimately involved in such factors as loss of

neurotransmitter receptor function (Schroeder, 1981). In this regard, it is

interesting to note that correlated striatal and motor behavioral deficits

similar to these have been reported in the present study have been reported

to occur as a function of aging. These experiments have indicated that there

are age-related deficits in balance, strength, and coordination (for review, see

Joseph et al., 1986) as assessed with some of the same tasks that were

utilized in the present experiments. Moreover, these alterations occ., in

concert with specific striatal changes such as decreases in tyrosine

hydroxylase activity (McGeer et al., 1977) and decreases in striatal D 2

(Joseph et al., 1978; Severson and Finch, 1980; Severson et al., 1982) and

rACh (Morin and Wasterlain, 1980; Strong et al., 1984) receptor

concentrations, as well as reduction of the muscarinic enhancement of the

K+-evoked release of DA (Joseph et al., in press).

While time-dependent assessments of the alterations in striatal D 2 and

roACh receptor concentrations in animals irradiated with 56Fe are presently

being carried out, and we cannot say the parallel extends to these

parameters, the present results provide, for the first time, strong evidence to

suggest that there are radiation-induced alterations in the nigrostriatal

system, an area that is one of the most sensitive to the ravages of time

(Joseph et al., 1986), and that these changes can occur at relatively low

radiation doses. A second area that appears to 'go gently into that good

night and does not rage against the dying of the light' is the hippocampus.

Numerous reports have indicated that this important 'working memory'

control area is both structurally (e.g., Brizzee and Ordy, 1979) and

functionally (Landfield, 1984) altered in senescence, and much like the

striatum is affected by HZE's. In this case Philpott and his associates (1985)

showed that morphological changes, which were assessed in mice at 6 and 12

months following exposure to 0.005 Gy or 0.5 Gy found that in the CA-1

area of the hippocampus, both the synaptic density and the synaptic spine

length were lower in the irradiated animals relative to the controls.

565

These experiments provide further support for the 'age-radiation

parallel' hypothesis which has been suggested in the literature for over 30

years (e.g., see Alexander, 1957; Upton, 1954). These early studies and later

ones have suggested that radiation may have a life shortening effect (e.g.,

Ainsworth et al., 1976) and may bring about changes in biochemical (Upton,

1954; Adelman and Britton, 1975; Aiyar and De, 1978) and cellular

(Ainsworth et al., 1976) parameters. Indeed, one study (De et al., 1983) has

indicated that radiation exposure ( 60Co source) resulted in enh.'nced

accumulation of lipofuscin (the 'age pigment') in the brains, hearts, and

intestines of the treated animals. Associated with these alterations were

increases in lipoperoxidation in liver as well as increases in the activities of

acid phosphatases and cathepsin in these same peripheral organs. All of

these changes are associated with free radical damage, are similar to those

which occur during aging, and are effectively prevented by dietary

supplementation with the antioxidant BHT. In light of the 'free radical

theory of aging' it is possible that chai1 ges induced by aging and those

induced by radiation may share a common chemical/biochemical mechanism.

Thus, while there are numerous, parametric experiments concerned with

dose, time, species, radiation source, etc., the results of earlier studies as

well as the present one indicate that under the right conditions accumulating

time in space can accelerate the aging process with some or all of its

attendant motor and cognitive problems.

ACKNOWLEDGMENTS

The authors wish to acknowledge the assistance of Drs. E. John

Ainsworth, Patricia Durbin, and Bernhard Ludewigt and the staff at the

Lawrence Berkeley Laboratory without whose help these studies could not

have been undertaken. This work was supported by the Armed Forces

Radiobiology Research Institute, Defense Nuclear Agency, under work unit

B4166. Views presented in this paper are those of the authors; no

endorsement by the Defense Nuclear Agency has been given or should be

inferred. Research was conducted according to the principles enunciated in

the 'Guide for the Care and Use of Laboratory Animals' prepared by the

Institute of Laboratory Animal Resources National Research Council.

566

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571

ANW6O FORCIS MAGIOSSOLOGYAISIAMICH IOTUT1

SCIENTIFIC18c ReORTR \D1 11I0" RISI NRi II 117, 185- IJ1989 SR89 -1 9

The Eff ect of -y Radiation on DNA Methylation

JOHN F KAIs I N(KH.' ioR(iF. N.CA[R \VS, AND S1I PH I N L. SNYDLR

Radion Biosheiuirt 1)cpzrmictit I, ou dl oes Radw~hioI, gi Rert 00/) iiiiiiiBr'iheda. 1lw inhmd2aU5 145

K si \i( IJ. F..(xt R-\\ s. (i. N.. \\i)S\\ iDR. S. he]F[([ci 0 Radiaion onDNNleths lation. Radual Rc' 1 17, 185-197 U989L.

1 he eliect of"'('o 1 radiation on DNA meth\ lation was studied in four cultured cell lines.In all cases a dose-deperoent decrease in 5-methslcvtosine was obsersed at 2-4. 48. and 72 hpostesxposure to 0.'-10 G.Nuclear DNA methxltransferase activx, decreased \khilc cvtoplas.mic acti'.it\ increased in irradiated (10 (Cx V'79A03 cells as compared to controls. No t)NAdemcths laseactisit% %%asdetected in the nuclei of control or irradiated V79A03 cells. A~dditioan-alk., radiation resulted in the differentiation ofG- 1300 NI [- II i cells, a mouse neuroblastomaline, in a dlose- and time-decpendent manner. These results are consistent with the hypothesisthat (I (genes ma\ he turned on IoL)loss ing radiation \ ia a mechanism involv ing hs pomcth\ lationolestIosine and (2) radiation-induced hspomcthvlation results from decreased intranuclear lcx-clx of tDNA methvl transferase. , i ss vademnc Press. Inc

iNTRODUCTION

The meth,.lat ion of DNA is a postreplicative process catal yzed by specific methyl-transferases which transfer the chemicallv active methyl group from S-adenosx imeth-ionine to carbon-S of a cstosine residue (/). DNA meth- ltransferases can catalyzetwo methvlation reactions on DNA:

I. de 0(11)1 methylation. involving methy lation of a cytosine residue on onestrand even though the cytosine on the complementary' strand is not methylated. and

2. maintenance methylation. in which the enzyme methylates a cytosine on onestrand using a meth,,lated cytosine on the other strand as a recognition signal.

Maintenance methylation constitutes the bulk of the methylation activity in vivo(2). In normal eukarvotic DNA. 5-methylcvtosine appears as the only, modified base(3) and then only in 5'-CpG-3' sequences. The nonrandom distribution of 5-methyl-c-xtosine in eukarvotic DNA and the fact that not all potentially methylatable cyto-stnes are methylated. along with the high metabolic cost of maintaining the methyla-tion pattern, suggest an important regulatory function for this modified base. At pres-ent the methslation of DNA has been shown to play a major role in the packagingand repair of DNA (4-6), the control of gene expression (7 8), carcinogenesis (9-

'Present address: Bjochermstrx Department, IUniversity of [exas Southwecstern Medical ('enter at Dal-las. 5323 Hlartr, Hines Blvd.. Dallas. FX 75235-9038.

185 00(133-7587/89 $3.00)(,,ps-ghl ( 14 h% Academic Press, Inc,\it rights mI renrixii,tion in anm form rescrsed

186 KALINICII. CATRAVAS. AND SNYD)ER

Mi. cellIulIar development a nd d Itl1erentiat ion( (12-14), and the stabilIizat ion of DNAconformational states (15). The occu-rr ,1 w e of 5-methvlcvtosine in DNA may alsoaloss the cell to discriminate between strands in mismatch repair (5' . 6). For example.damage to the 5-methslcv tosine residue of DNA. resulting in deamnination to formths mine, could be correct ed by the appropriate repair enzymes using the undamagedmethxlated DNA strand as, a template for repair. Methylated DNA also aids in thepackaging of the D)NA into nuIcleosomes. It has been shown that DNA containing 5-meth\ Ic\ tosi nc Ps packaged into nucleosomes (4) and is located primarily in the coreparticles (11)

livpornethslation of DNA can result in activation of normally quiescent genes.Glenes shoss ingnmethyltilon-induced deactivation include hamster adenuine phospho-ribos\ Itransferase (S). fleLa cell and mouse hypoxanthine phosphoribos\ ltranseraseI(- 1,1). and metallothionein (19). Most of the methvlatable gene sequences impor-

tant for determining expression are in the 5'-regions. Whether 5-methylcytosine pre-v ents binding of the proteins needed for transcription or allows binding of repressorproteins is not s et knowkn.

I, 'Itra\ iolet radiation exposure results in a decrease in DNA meth\ lation (20-23).\pre\vious stud\ i n'estigating 1 -radiation effects on DNA methylation in rat thymus

and hone mrarrow% in vivo (24) provided no clear-cot conclusion on the effiect of -radiation on DNA. miethvlation. In view of the important role DNA methxlationappears to plax in reguhlting gene expression and cell funclion. this study w as under-taken to insestigate the elffect of ' radiation on DNA methylation and its possiblerelationship to gene induction. It was also of Interest to determine the effect of -radiation on intracellular distribution of DNA~ methyltransferase activity.

N5IIRIALS XND Mi tIHODS

( It// II I/-r (

Nutrient %Mixture 1.-12. minimal essential medium. Lagle's minimal essential medium ssith Earle'ssalt,' tulbeecosN modification 01 Farle's minimal essential med~um, fetal calf'serum. penicillin. streptoms-, in. gentamdi;. and I-glutainine %%ere purchased from (iIBCO Labs (Grand Island. NY). .\-2-Hsdroxv-eth I-pipcri,'ine- \ -2-ethane-sulfonic acid I tiepes) and dimeth\ lsulfoxide (DM50O) \kcre obtained from theSILema C hernICa) ( ompan\ (St. Ilouis. NlO).

(hiese hamster o% ar\ (CI 10) clone K- I "sas obtained from the N5merican I %pe Culture (CollectionIRocks ille. Mil)) and A~as maintained in Nutrient Mixture F-I 12 supplemented Aith 10l(; fetal calf serum.10(0 unIts ml1 penikillin. 1001 mg/mI streptomscin. and 210 pg/mI gentamicin.

Chinese hamster lung fibroblast I V79J) clone A01 was the kind gilt of tr. tomn Walden. AFRRI. It wAasmnaintainedl In, minimal %essetial medium supplemented \kith 10'; fetal calfserum. 1001 units/mI penicil-in. 10 ((t g'ml streptomscin. 210 Pg/mI gentamicin. and 2 m.11 I -glutamine. T he medium \sas huticed with

S mM Ilepes.Hl a clone S-3 \kas, obtained from the Amenecan IType Culture Collection. It \kas maintained in M/3

medium M tr. l'inhas F uchs. personal communication). M/3 medium consisted of 1/3 vol Fagle's minimalessential mediiim Asith Iare 1ats 1 sil Nutreiit M,,isture F- 12. and 1/3 %ol tDulbeccos% modificationof' Farles% minimal essential medium supplemented ssith 10'; fetal calf serum. l100 units/ml penicillin. 100,vg nil streptomci n. and 21) gg/mIl gentaamicin.

\touse neurohlastoma -1 300 clone N I L- 115 %4as the generous gift of D~r. tDas d Krause of the 1/ni-lorined Sers ices I 'niersits of the Ilecaltii Sciences (Bethesda. MD) and was maintained in M/1 medium.

\1l cell lines sscre maintained in minolaser at 17V in a humidified *;. (0-,/95'; air incubator. Themedium on irradiated cells, %as, changed dail% ito remose dead cells from the Culture. Di)tkrentiation ofmnouse neuroblastoina cells ssas accomplished b\ adding dimeth\lsulfoside to 2" and incubating lbor 2

t)N- N NIIHl A ION 18-1

djass at ; 7'( .Phase contrast micrographs of(iiemsa-sta ned neurohastomna cells wsere taken with a ZeissN \i0 OPhOtOmicroscope.

Irra1diation ss as pertOrtued hilateralls at room tempet ature using thle AFRRI 'Co -,- radiation source\kith a dose rate Of I (6s n11n11 1 otal doses rangitle fromt (ito) (is %%ere gisen.

OOOMMoioszti 50.- Ilt!(Iii/( Il"%t I''J el

(ellular D's .\ as isolated using a modification of the method of' Rim and Statford (25, 21 fhe D)NA,%%as hs , drols/ed to )Is deoss rihonucleotides dN.MPI l6Illo\ ing the en/s niatic method of ( hritstman -.Separation of [) \ NdNNIPs h% high-pressure liquid ehromatographs sas perfoirmed using a BeckmanModel 344 gradient 1liquid chromlatograph s ithl an Alte\ liltrasphere -m particle octadecs Isilane column4.6 mnm vd. - 2i cm. Samples, %\ere eluted ssith l1l0 m t1 sodium phosphate buffer. pi1 55 oser ~'min

st amibent temperature s\ ith a tlos rate oif I mnlimin (2>) Detection ss as at 254 nmi ssithi a detector senlitis -

its ofi).(15 ahsorhaitce units full scale. Data \%ere anal\sied wkith an IBMJ Ssstem-9l0l computer interlacedto the I I'Cssteie. 1)eo\\ rihonuclec *d: st_- i:-s %%ere purchased from the Sigma Chemical Co.

lermination of acets Icholinesterase actis its in cell homogenates ofnmouse neurohlastoina cells sasperfoirm ed usi ng thle mnethod of 1.11man cliai, (2N). Thlle cells "~ere harsested h\ fIircefulIs pipetting astre amof mledurn oxer themn. -The cell suspension \Nas centrifuged at Xlllgand 4V 61, 10 min. [hiepelletwsasssashed once skith Hlanks halanced salt solution (IiBSSI and once wkith 1l00 ml sodium phosphate huller.pff I 5(1. resuspended in 21 ml of phosphate hulfer, and homogenized b' " %) strokes in a Dounee homoge-ni/er. I-lie re.-dion mixture consisted of' 2.61) ml of' 10011(1 .,!)~dium phosphate buffe~r. PH1 8.0). (1.410 nil ofcell suspension. 0. 10 til of'dithiohisnitrohenioic ac:id IWalhiochem Biochemicals/Behring Diagnostics. L~aJolla. C. atid (1.02 til of acet% lthioeholi ne iodide (Sigma) w hich swas added to initiate the reaction. Theahsorhance at 412 nni ssas ieeorded for inin. tisits ssas determined using the change in absorhanceper ininute os er a linear portion of the eurse.

PI, t'im /)i'h'ro11iioi w

I he miethod of' c i- ssr alii (29) ssas used to measure priotein content.

I 'i' I Ih~iiiiaihri .Iiri

N1 13 single-strimcded D)NA. %I113 I 7-hase primer. h co/i DNA polsmerase 1. and -14 ligase were pur-chased from BR! (Bethesda. %11)). A I P. dATP. dTT P. dCTP.R and dOiTPssere obtained from Bochningr-Mlannheim Biochemicals, (Indianapolis. IN). 5-Meths-dCTFP ssas purchased from Pharmacia (Piscata-s a s. N I).

I he method of S/sf ii al (30 whsas used to Prepare unmethylated and hcmimethslated D)NA. Douhle-stranded DN\ %sas ss nthesized ill tiaro using hacteriophage M 13 single-stranded DNA as a template. Tem-plate f)N\% 10pg) as incubated for 211 min at 15'C~xith 0.75pugoui 1 1 7-hase primer. After incubationthe liillossing wsere added in a final reaction solume of 3111 pl: dithiothreitol. (I10 m.1f), I ris-HCI.1 (pHf _.4.W, mnit!). %Mg( I (6.6 m it). NIP1 (0. 1 ml!M, F co/i DNA. polymerase I (large Fragment. 7 units). 14 ligase(401) units). d\A [P )50p if. (IT [P 50) pA!). and dG.IPO 15(hll For unmethslated DNA. dC1 Pswas addtdto i)) p l/. Iior herniineths hated D)NA. 5-methsl-d( '1 P was added to 5)0 P.M. [ he misture \kas incubated atI fo lr I S h and the reaction \&as terminated h\ the addition of ED'I A iii a fitnal concentration of '1) ml!.

I: i/'o, //on ,I DA It/'i/ii/io'

I he cell monolaser "sas "ashed once with 1HBSS and thle nuclei wsere isolated using thle Butler method(3/ . T he nuclei wsere pelleted h's centrifugation for I5 min at 800,if and 4TC. IThe supernatant ssas sased

and used to determine estiipla.smiC DNA mithsltransfeiase actisits. [ile nuclei sseie washed twice %kith

188K \1 INICI. (C I R \S 5. \ND SsN~ DER

8-

7-

6 -

5-

5-Methyl- 4-cytosine

3

2- 24 Hr Post

1 72 Hr Post48 Hr Post

00 1 2 3 4 5 6 7 8 9 10

Dose (Gy)

I-a 1. 1.-Nlethx cx tosine chanpcs in -,-"'Co irradiated C'HO K-I cells. Cells scre exposed to 1. 3. 5.orM j of radiation. \t 24. 48. or 72 h postirradiation. the cells wre hars ested. the D)NA ssas isolated and

fixdroicd and the resulting nuLCleotidecs "e re separated h\ H PLC. Dita points, are the as crage of nine'Icterminations, IErrorh bai rc gis en in standard de\ iation.

HBSS. then resuspended in 1.; ml ofT ris-1CI. pH 7.6. containing I mrl El)! S and I rn./ dithiothr ,itol_IeI nuclei %%ere disrupted hx 3 s of' sonication after \Nhich the Mixture "sas assased for nuclear DNA,^

m~cth\ Itratisterase act i\ it%.

[he method ot*f ,st / 1301 Asas used to assa\ for both i/c'nma and miatietlancenmeths lation activit\.1 he enl,%mc assas% mixture contained, in a final kolume of 25 4i. (1.3 Ag of" DNA lunmeths kited for dp'ti o meths lation dleterrmination. hemimeth\ fated for maintenance meths lation determination)1. 16 PAIS-adcnos\ l-t +[ncth i-lI methionine (1(5 mCi/mmol: Amersham. Nrlintgton I leights. IL). and 3 Al of en-/sine mixture in a buffer containing 20 millris-HCI. pH 7.4. 251' glycerol (\ /\ ). 10 m.11 EDIA. 11.2 m.11l'\SF. and 201 m.11 2-mercaptoethanol. The mixture wkas incubated at 37'(' for 6(1 min. The reaction wasterminated b\ the addition of' 111(14 of Ill' sodium dodecvl sulfate and 100,u of'I NNaOH and incubatedfor an additional 2 h at 60'C. T he mixture "sas extracted with I vol ofa 1: 1 mixture of ehlorolokrm:isoamslalcohol. The lax ers wkere separated and I \ol of' l0' trichloroacetic acid (ICS) wsas added to the aqueouslaser. [he mixture %kas left on ice for I S min after wkhich the precipitate wNas collected on a Whatman GE!C glass microfiber filter. Thetfilterwkas\A~ashed tw&ice sith 10"; ICAcontaining 1001 mA!I sodium psrophos-phatc. wished twice with 711*; ethanol, dried, and placed in a scintillation vial: 5 ml of'a liquid scintillationcounting fluid ".as added, and the incorporation of radioactivity was determined b\ liquid scintillationcounting.

RIESU I S

The etfect of various doses oft-y radiation on the percentage ol 5-nmethvl cytosine incellular DNA is summarized in Figs. 1-4. (CH-O K-I., HeLa S-3, ('-1300 N IE-!1 15.and V79A03 cell litnes irradiated with 1. 3. 5. or It) G,. were harvested 24. 48. or 72h postirradiation and the DNA was isolated and hydrolyzed. [ollowing HPLC. thep.~rcentage of 5-mnethvlcytosine w&as calculated and the distribution in irradiated cellswas compared to that of unirradiated control cells.

l)\\ Nil FB"l \llON\ 189

cyto sine

24 Hr Post

- 72 Hr Post

Dose (Gyt

I 5-lethJc~tsinechanges inll-' o irradiated licla S-3 cels. (ells 55etc e\pkscd it) 1.1, 5. orH) -,t radiation. \t 24. 48. or '2h postirradiation. the cells kserc liars ested. the DN N \s al ilatedIC nd

1ix droo 1\i. aT1d the resuhlill1 nucleotidecs ss crc sepa rated h\ 1111 Ci ( a ta pomI iiO rc the as eraie Of ninedeteIrinaioiio I rror hairs ate i!]s en in saandiard des iation.

As shoi.. II in lIg. 1, 5-rneth\ lcx tosine decreased in (1 J 1K- I cells ffllow\ing radia-lion) esposure. I he prrihahilit,. based on Student's I test, that the numbers are notsignihicantl\ ditfcrent wxas P' -- 0.002 In all cases.

licur 2 shows the ele'ct ol radiation on 5-rnethkk Icuosine levels in Hel-a S-3 cells.Again there is a decrease in >-methv lc\ tosine Iecels at all radiation doses and timesW- <0.001i. As xsih\t ('1k) K-1 cells. fil~a S-3 cells cxhihit dose-dependent. decreasesin l-cl' c ostic at 48 and 72 1h piostirradiation. Iliowexer. from I to 5 Q, a plateauis seen at 24 h1 postirradiation.

6-

5-

4-

c ytosirte

2-

1 24 Hr Post48 Hr Post72 Hr Post

00 1 2 3 4 5 6 7 8 9 10

Dose (Gy)

I N, 5 ltskt iecagsi -, -'"(o irradiated V79-Nit clls(Isser xoe tk1 .3, i. oir Ili

(6% itiradiation \f .24. -15. or 71 ft pistiratiatitti. titcel m1% r harsested. the tD\ \ ssas isolated andhx droix ed. itiki The resultinig niuelcitidesN ssc separated hs 1i P1 ( D ata put its are the as erage kit litte

determititins I rroir hars arc gis n in stattdarkdcdes alin

19 K \1 tNI(It. (Ai R\N\ %S. \M) SN) I tR

cytosifle 24 Hr Post

72 H r Post

Dose (Gy)

I i 4 -oilol\itot atgoin -2( o irraltated ( -1 300( N I 1-1 1 'eel). ( elk vcre e\iowod to1,. 3If otIt (i j ot radiatioon. \t 24. 48. or '2 ht pootirradiatoot the eelk \kcrv Itarxesied. tile DN\A xioas Isolated

.ind hdroI\m'd. and the rtLti ot1g MiCtJLC eetiew oreparated h\ 111'1 D.I ata poiti mre the as erage oflttotedetrttotaiooisI rroor hzirsare ein it it staitdard dlei labon,

I he etli~'c of', radiation onl 5-rncth\ Ic\vtosine lev els jn V79A03 cells is shoki n inFiW. 3. A dose-dependent decrease of 5-meths lcstosine in irradiated cells is also ob-servedc: hoxmever. in this case the dose-dependent relationship is seen at all three timespostirradiation. There is no I - to 5-G\ plateau region at 214 11 ais seen inl HeL~a S-3.clls. Sii-niicance "as at thle PI- 0.001 levc! in all hut ftOur cases. [hle most importantof these are the l-Gy\ values at 24. 48. and 72 h. At 24 and 48 h P' -(0,00l. but at 7211 1) - 0. 1, indicating that this value is not signilicantlv dlifferent f-rm the control.

Fhe percentage of 5-rnethvlcvtosine in C-I 300) N I F- I I S cells is shomxn in Fig. 4. Adose-dependeni decrease in 5-methl~cxtosine is seen ait all three posti rradiation timlesinvestigated. I , aill hut two cases. 14 and 72 h at I (iv. the percentage S-methvlcvto-sine %%as significantls decreased WI- 0.001 ) relative to the controls.

Experiments wre performed to investigate thle possibilit\ that the decrease in S-rneth\ lcvtosi ne lb lowing radiation might be related to changes in thle intranluclearmecth\ ,ltranstkrase (Nil) activt\. V79AI)3 cells were irradiated wxith 10) (0, of'-, radia-tion and harwestcd 214. 48. and 72 h posteXpoSUre. Maintenance and (It, novo Nilactix ities wecre assayed in the nucleus and the cytoplasm if othe cells. -tables I and 11sh~ the do' roio and maintenance DNA rneth\ ltransf~rase activit (if control andirradiated coils expressed as picornoles of 01-meth\l groups Incorporated into D)NAper 601 m1in incubation at 370 (Vmg of protein.

Ft llowing radiat ion there was a redistribution of' both nmai ntenance anrd (It no lmcthx ltranslierase acti'~it\ fromn the nuclear to the cytoplasmic compartments. In thlecase of'/(, lovo Ni activit\. a decrease in thle nucleus was apparent b\ 24 T,. at whdichtime th 2rc was a drop from 0.36 to 0. 14 pmnol triti urn incoirporated per hour ( I ableI) 1 hi-. (10 case \%as paralleled b\, a concomitant increase in cvtoplasmic, dc' mo

D)NA %IF I IIYLATID.N 191

[ABLE I

D(~ ' DNA NMethslItransterase A\ct iits in V79A03 Cel Is

Control /0 0'1

('05~~~~ I ,00 SD)0 011

N uclear adels It\'4 ) o, 6 0(6 0.6 [9 0. 14 0.02 2.3 (0.1

W1- 0.00 )

-2 h o.321 o.03 4.9 0[4 0. I S 0).(1 '.2 0.2W.' 0(.001I)

(toplarmc actls it\2401 0..0'(3 4,8 0(.3 ().78 0(.02 12.4 *0.4

(1' 00 148 h 0.28 0.0 3 4.3 ((,5 01.4 ((.04 8.9 -(0.6

72 h 0.1 ' ((((0 2 .6 0.Y 1( ((.03 ,, .. (7

(P- 0.10()

X,'u Data arc the mean ot'si \ determ ination% and are presented i n acti% it% units. One u nit ol dclix itN isdefined as, the incorporation of I pniol ofl H frorn .- adenoss (methionine into ON.A %n hen the reaction isi ncuhaled at '3? '(' (or 6(( min. E rrors are gi s en in standard de% iation (SID). Significance \% as determinedusing Student's t test.

methylation acti'Iitx from 0.31 to 0.78 pmol tritium incorporated per hour at 24 hpostexposure. The tic novo MT component found in the nucleus of irradiated cellsremained depressed bN approximatel\ 50"; relative to the controls during the 72-hpertod studied. Nuclear maintenance NIT activit0, which accounts tor approximately65';1 of the en/s me found within control cells, also decreased followIng radiationexposure, but in this case a significant decrease "as not seen until 48 h postexposure(Table 11). Once again the decrease in nuclear MT was accompanied by a comparableincrease in cytoplasmnic MT (fIable 11). During the course of these experiments there%%as also a decrease in the total cellular MT activ ity (di, e ml plus maintenance) fol-lowing irradiation. For example. over the 72-h period investigated the total MT activ-itv found in control cells remained constant at 6.4-6.6 pmol 'H1 incorporated perhour wkhile the NIT in cells 72 h postirradiation had dropped to 4.7 pmol 'H~ incorpo-rated per hour.

It cl Iit hoilleI/tetra~w Indut1imn

Figure 5 shows the etlecct of radiation osn acet\ Icholi nesterase activIt,. inI C- 1 100)N I [-1I15 . a mouse neuroblastonia cell l ie. Acets, Icholinesterase is a miarker for cellu-lar dlktrentiation i n C- 1300) N I [-115 cells. Specific en/v\ me activIt\ \\ as determinedin moles o~f-substrate hvdrols,'ed per minute per milligram of protinil. Thle results inFig. 5 are given as percentage of control. Cells were irradiated with 0.5. 1, 3. 5, 7. or

42 K\ Ifl( !1. (N 1k 1 \ \k \\) S\) Dl) R

I NI1)1 I II

Ma~ii teince I)\ \ Ncth1i hr , i',- \CtILt\ in e 9\() ( clk'

( , r 1,) Iutf,

24 h 4 2 ) 4q ! ,4(t\

0)' 1)14, - 1 Is S, *' --

22 € ,

I/'. I 11)1I

-2h 422.~ M. ',, i - 'I2so(Il l 4s.2 24

-4I I4 IlS_24 2 I 42 () O'l -2 14I' ((1.

41 4 I.() (o2 . . .()2 4.,4,'+2 4 ",w "4, )I)

'' 11 ))-"1h I.-h • 1 l 2 .2 - 2.1 2.13 1 ).l1' 45.)) 3.5

I' (.0) 1

N it a I al r the nlcan o'\ dctcrlmnatior mnd aire prc lted i Ol acti\li\ UmKl II(nc unui of aciiN' lt is

d hlincd ai the ilcorpor.oIIl (, I I pilll of I I fromn .A-adklno s Ini f(lionin info D -A "Mfien Ifc ro1 I l'lli isMinuhted At ,'( tor i) ilin. Irrors ore gi.cn in standard de\ ltion I(S)). Signilicance \\as dternimdtl",IFIK Slu,..cnt", I tOMt

IMI (G', of' radiation. har estcd 1. 6. 24. 48. and 72 h postirradiation. and assayed foracct\ lcholincstcrase activit\. At I h postirradiation. enz,,me activiit leels were at orbhelos the controls. Activitics rose at 6 h postirradiation. but the increases ,cre notsignilicant, above controls, except for the 7- and l()-Gy points. At 24 h postirradia-tion all doses resulted in acct\lcholinesterase levels greater than controls. VerN littlechange was seen at 48 h postirradiation over 24 h except for 10 G\ which increasedfrom I 50 to 191'; of control. The results obtained 72 h after radiation exposureshowed increased en\ me activit\ over control at all doses in a dose-dependent man-ner. Ihe greatest increase in activity was seen at 10 (iv 72 h postirradiation where

acet\ lcholinesterase activit\ was 234"; of control. This compares to an acetylcholin-esterase activit\ 244' of control in neuroblastoma cells which had been diflerentiatedwith I)MSO.

To determine if the i-wreasc in acetylcholinesterasc activity was accompanied bymorphological changes associated with dittfrentiation. photomicrographs of control(unditerentiated). )MNSOdifl.rentiated. and irradiated (10 G',. 72 h postirradiation)ncuroblastoma cells were taken. Figure 6 shows that both the DMSO-treated andirradiated cells exhibit axons indicative of differentiation in this cell line.

)IS(CU. SSION

(ene activation and inactivation, strand selection for repair. cellular diflierentia-lion. conthrmational transitions, and carcinogenesis are just a few of the processes in

250

200-

of 150 24 Hr Post

Control

6 Hr Post100-

Dose (Gy)

1,1~cls (- Iill) il I iccisflrXNJlrL] tlSCS I -, tla ti \ Ctc t1t1t.StI.\l '11 Ilic Itikfildct

it, Iiji si'3~4 of citrol. Daita points '1 tc 'i\tAcI*.Igc of nm11C (ICtCrtltifldtlTIN It harN are tLtsc1 III

'I lli sic 'at ioT

xx hich M )*\ flictl~ation arnd 5-rncthx cxtosine hax c heen Implicated. Ihfe delcteri-ohIII' Icts of radl(iationi arc x c ll k noxx .but thc mIchiZtlSml bx wIch damlage is trans-m ltIcd to thc Cell and thc exact nature of the radiation target xi thin the cell are stilllal-11Cl u nlkno\x n. Sinrce DNA *\ mthx ation and 5-mecthx lcviosinc appear to plax anlImportant role In ceeinduIction and DNA repair. \w inritiated this project to assessthe cfhcet of'-, radiation onl 1N \ micthxlation.

In the preI-sent Studx 5-mlCthx lCxtosine levels were found to decrease in a dose-dc-pentdent man ncr n clt tIred eellIs receiving up ito 1(0 G\ ""Co , radiat ion. Il his de-crecase xxas seen at 214. 48. and 72 h postirradiation. [he greatest decrease w~as seen atIM C1\ an 8 h posti rradiatiori in aill eel Is(Figs. 1-4). At 24 hi there is a plateau region

arudI to G in i(r'1 IC K-I and HeIca S-3 cells and 5S to 101 (i\ In C- 1300( N I FI§Cells,. [ his alpears to be a tissuec-specific phenomenon since a plateau region is

,,en at I to S (ix in ('111 K-I and Itel .a S-3 cells. both ol xx hieb are epithelial lines.I[he pllateaui region is riot seen uintil 5 to If) (i\ 'in C-1I3001 N I F-Il 1 cells, a neuroblas-tomla ti tic, arnd does riot appear at all in V 79A03 cells, a fibroblast line. At 72 h a,,light increase rn 5>rietl\cx tosine levels over 48 11 vaIlues .vas obserxed. possiblxIndicating the beginrini ng of recoxerx.

Deccreases in *;-mcthx\ lcxtosirie content in D)NA earn result in the actix ation of nor-tuallx quiescent gi-nes) ( . IS. /9). 1 kxporiielhxlation ol'[DN '\ has also been correlatedxA iti celular dittlcrentiatiori (13. 14). (C- 13011\N I F- I I5 cells, a riiou.se neurohlastonaine Used inl thts SIudI\. are maintained rn culture In a nondrflereritiated state hut can

he Induced to ditlkrcnltiate lix exposure to a chemnical stimu1111lus Or \ rax s (32-36).

(pon dificireritiatiori tie cells exhibit awons (36) arid prtduICe aectxlholinesterase13.I :sing aeetx leliolinesterase ats an en/.\ me mnarker of cellular dilfereritiation. the

clicet of -, radiation oil ('- I1300( N I L- I115 cells was studied. Cells harvecsted 24. 48. and

194 K*\ \l NCI. C% R X% %S. AND) S\Yt)I R

li(,. (v. Phase contrast photomicrographs of (iiernsa-stained G 1300) NIF-) 115 cells (SOO - magnifica-Ho) a ( onirol (unirradiated) cells. Nh DMSO-treated cells (2 7 DMSO/2 das s). Mc Irradiated cells) (10

6N,1 72 h post). Both DMVSO-treated and irradiated cell% exhibit axon formation indicative ot'differentia-tion,

72 h postirradiation show dose- and time-dependent increases in acety lcholinesteraseacti% it',. It appears that. at I and 6 h postirradiation. the cells have not recovered fromthe radiation damage. Since radiation causes short-term inhibition of hulk proteinss nthesis (3S~), it mnay take up to 24 h postirradiation for the cells to express the prod-ucts ot the newly activated genes. Additionally, irradiated cells ( 10 Gy, 72 h postirra-diation) are morphologically similar to DMISO-ditkerentiated cells (Fig. 6). -1 hus ourresults shoss that radiation-induced hypomnethylation maN lead to diflfrentiation of'mouse neurohiastoma cells as indicated bN increases in acctvlIcholinesterase activityNand morphological changes (axon extension).

Inlaill it \\,a" of nitcrcst ito k no% isshcthcr thc obscr\ cd changes III thilemt\ ctosilnc con tcni 0) I)N \ canl he related it) chiantws InI n ICIir )N' -\ N11 actti\. M Iain-

tCnallk: nc 1 I IactI it\ II tile n IClci of rra- djatCd cells dccrcascd to appri inatcl\ N),0otrol - at 4S and _2 11 postliradiationl( li 1). 1Ihis, dccrcasc Is paralilccd h\ aonicom tla nt Increase InI c\I top,lm Mc antcac I IC\ ct\ 4S andl 7 2h p stcs po-sure. ( hi ta , k also % J a ma-KCdI r-cdistrihIon11 o1 t' I/ I I I,"M I act I\ It IrIm IIhclin uclcar ITo thc c,, toplasm ic coin lpartint 10 Ilo%\ inct- radiat ionl I hlCsC rcsnIlts nd1(ica'tcthat 1)N \ A lk) filetl 1 1aI Io n t~o Is ic radiation rcsU)Ilt l in largc part. fromn radi-atIIin-Ii, cc I -L eiIcakagc olt N I I from11 the I) ulcuI Ls to tile k,' tOplaIsin. AI ltcrnat I\ cI k. radi atI in

damao1!c to thcile ca ILIcns clop0Ic and"O ' o ' 111,c copl ii ipai file ransport (0,n~css I ss nthcsi/cd \I I Into thc ucLcuCs, \AlthouMd ai dc mIcthslasc acti11 11,its hbccnlreported (-(). \\c I'onnd no cs idcncc SIgIccst inc that CrI at ion1-i nd nccd Ix oc at in Is IC c to thnilIL uct Io ol 1' a( dcmIncths\ lasc data nokt shloss 111

\Inot hcr 11ictmr that maxI r-csuIlt InI hx\ p~omcth 5 lation aftcr rad at ion Is Inicreased Ics -

cls of an \I I Inhibitor. *\prelimiarx r'cport onl a natiurals owcnrrincu inhiblitor o)ID-N \.%I I haZs r-cccntl bcc pub[1[LIIishcd 4o. 411. 11I:11]1 I hcX iniitr a ITTs inlccnlar1 \\ ciglItaiinic called mictliiin \\as oricinallx isolatcd fr Ioml rabbit Ii" cr. bUt alsoIII habccntOLInd Inl hmncrx thlOcukcmila Cells. \lthouch41 it Is p)osSibClc hx pomcths lationI of'1)\ \ inl irrad iatcd cclsT coulil r-csuIt fromIl InlcthlI i ni n ihitionl oftli ni ctsIt ra nslI'CraIsc. onII rVLI rcslt arc 1- mocmosisicts\\itll a rcdistri bionI1 of aIctiI its0fro thi 1IICLusto w c\ o plasm llossI' ig iadiati ncps c

\\c hIas c dcmlonistratcd thlat -,radl1I11 iaionitcrIl-CS ss lil th l t 1w mct h ltionl Of I \and that tilc act] i at ion of ccr-tai n gcncs canl als;o occii rflls '1\ nu 1 ra idl atonl C\pOSiIi-c.A1111 1 It ~ f ho\t ou sidcnIcc is 01n1 xsu ggcsti Sc. It sccmls rcasonablIc to )ostLaktc that t hcscphIcn)omi~l~ nain\ bc iclatcd. R.ccntlx It has becn shoss n that onlcogcnics canl bc Inl-dUCCd b\ u Tlin ii u1 ril ation 1421. Whcthcr thcrc: is a causa1,l ratinhipctscnradlation1-iiidJUCCdI1%10C hx poc h loll. 0"oIcuIccn (induction, and tIinon'ciics'is rciains"Unidctcrmi ncd.

Rt ( I I\ ItI): Noscibcr -15 '98 71 RI % IMtt: %larch 16(. IL)SS

Rttll RIM \( [

M, 6t1 1~ ). j [it R\55 I/. and MNi. \i s. I lie en/\lic nmcilil~aitm )t R\ \ and D's \ It I In iho:Pccics '4pccltwll\ '11tihe ImbohIatian Pnsnsi', Vt! i, ad 's' I N 1 50. 16 169 a

I s I \ Ii I N I U t' *\ 1 . .6 vs .t \ N niCihslanan1 ill 1IM n11all,11 n10,10 86 w, t 24.

SR . 1) I il 55 I lie quaudtiI c sCJIAaaika at pnrncs. [)% rnn111(tiC'. and MAci 0IdiJC 11% ii 5)T

Jironhaigraphx. .1 11,' hem~ii 17-5.31W ( 1944 R. I ,' 11 \i s. I -I , %1 K Is - . Dt i , . and R. t1Il RI [()\,~ \lCIhxlaiin1 Of MsLICa 1I JIl a d

intClcasc cflsiti\c I)N \~ h id,! Rc' 4. Ik-ilI1. 1 11 siRi And t. 1I, 1 \) I (W. ()IC 101c 6r M)\ nihxlatin in wcrichialt scik is strand dsiniina.

11)11 inl mnl atch repail rI,), \at/ 1I)! S, i .I S1M ,-1. -- N ( P),";)MI 14)m. s. R, %\ \(,Ni i. and~ Ni Roi )i \\'. Nlisati ri-lIIll 01 &XMMnaid -lsIctsleiIl

16'! 194. I, 5 '1 l'JsII-'I \ ss i-NI \N anid It. ( i I D\R. \Ctisc guile SCLiI'iCI.1C LarcunInobs0~ iIS' \Si!f Il o' SI

I N 1 78. 424t, 4(1 11 i I )sI I,15 . K i sin i . -1. N isu \ i II and tI, (i .sI livori iregional I )\ L' - IT\ Iaiirin MIn geneC C\ isl C I'r P

Nall tad s, ( t5 A82. 2 6(-- 564 I 198 551,

19 K \1 INl(H. (\I R N\\AS. \\D) SNN DIMR

I , j B('1 ii\i and D) D)R \11 It ',KN lit .lnlcihtlation of' I)N\ lin Ran cell' after treatment '.tith A-meth'. - \-Ititro'.tiurea ( .o , 'einiui,01 2. 39-42 ( 1981

11) S. S. fit( ii -. N I iti ,.III'.\. AC ( \'.'.~ \N , 'i and N Ri'. i as4)\.( Cntparati'.e tunoirigenicit\ and[)N .\ methtlation Ii I 344 rat'. b\ - ittnio.mnll i

3p.rd.hI-uatn and .\-nitroi-

'.adinieth'.laninic ( i- P' R,'\ 46. 498),-5i1980)tu.

11 1 .1 1 1 \ it . R BHi it'.nd R. \\ I Rill ,lDecreas.ed nieth'. juan rate'.f aD NA Ii S\ -4i transitirmed

human fihr hlai't ( aih p' 57, -(4--(iS ( l'iSOuI

12 .1. K. ( IIRIN.I V. \N. \. \t It I (11 B, Si iiil NBIFR %1. SS. S( l~ 11 )I RIiA. anti 6 . Nt S.. I k ptneth\ latinof )A -\ durintidiik'renitiatiOn 0iiA ricrd crthroileukenia cell'.. ./ ( ,// 11,l 86, h-3i I lYlIl.

\1 NI. Si I III R. R . Rn IiN I. and P, XN. N1i 'R K',. l, Mrine0''- rxi'ICU k: tii cI:,-,!2 Jtf-.rri dt it)i. I) N.cI h% le r.e n'.it i I\ An11d I1 N meth\ laton near the glohin gene'.. it,(,il NIt ad Ni I N. 179, 1 I SO-1 184 1 N'12

14 \. R'\/I\.(C. \k I mi. M. S/ I . J. ) i'.R\[iII . RostI iti'i %I. I N \\I iAI \-N . N. St i \K)-(r.II IitI.andi 11 ( I )D.R. %ailAIMnIN IN DN \nteih\ latitin during mau'.e c ell diikrentiation1 InI it1\ and lil

15 Ni \1i lii and G I I is \.I i i i), Itkfco'..tiet'llanina'.ihei i nu6LcleOtide: tihe BI-/ tranISItion

Inpo (t~td( 1-11( I;-piit(d( a-fi. Iraiw \all ha.'Ni t '. 178.tiI1123 iI.

let "El \ I 17.~

I R I'. \KH and . \. \l()Rki,. Ncti'.ation ala ntinewircsed iI\poxantilrptihc rhi. trn.ea,

alleCic InI 11titant H-", Ile[ a MSl' h'. agein' that inhibit D)NA mcthtlation. 1/. / hit,'! 6. 9--.W4 iI 'i)SiI,

I I x 1 ISK I ) %\. Ni I N I . I . \ t'K anti ( i. R. Ni \Kt I i\. NMth\ latimn of? the buut hpri genec

di tier' Oin tile act't\ C andi nat It\ c \cit i ornne.. I/,,/ ( El /1!,, 6,'It 1!44 '12'4 ( I 10I.

N' S, .1 ( C. \i'i Ri and R. 1).) I'I i Rt. I) N \ tch\lation control' tice in~duIcihilit' ii thle M1USd nMetal-

loithionci n-l LUcti Ini 1\ mphid cl'.( .11 25. '3 -240I 1 NS 11.

\1 N I ii'm . I., I . Rt \1 i. an1d I . BE tkIt K. Nilth\ lation oft )'s N in Ilel a MIS altir itilra itilet Irradiatlion.

l1w ./ Radial On,,'I i'! /'/it , I. 2s'i-'14

2/ i 0 '.'. sand I . Si (,so its. lihhition otI)1A.\ nctl~alion and radiation Hinducti1on at nrne ndii-

eetlu' t irute.. .1 Raa R'. 23. ; I 191521.

\1 NI. V. I it iiiit Ri \N. I . R. Hi mi iI. and R. I ). 1I' si t I it., I ltraatelt radiatioln-Hiduced TIntallothiti-

TIlilI-! cLICTI acltiMinI IS. aStOCIated %k.ith etensit I)N N denieth\ lattoit ( c1/ 35. i-2141(1 9S i. 1

23 I- I lit i Ki Kt. F'. 11 IS.i. NI. RI t HuR It 5 and J. 'S. I \I) RiI 'ertuhatin at ntaillterarice atnd

dc nota D, \ mnch\ lalion Ii \ Ira0 h\ I NH 'i4111111 IILItdtlt p\ imidie photiidiniert Pr,(\,ill 1, ad Sj / IN 182. 01i.1ilR I 191,";.

24I N R \Ki i\ \. I'o't-riadiation nietlit latitin otdc rim o '.t tieed~ I )N \ of tat htone mnarrott and tttt-

BI Hi I nd 1) Vt . SI \ sIi ()RiD. N iettera'l Medthod lt the isolation Ai high ntticlar t.cigt l)NNfront euikarwaie'.. \No, Ii, li,!. RE , 3. 2303-2301197 l,1.

( old Sprung I larhor I ibolrator\. 191.

J K. Ci Ito I \1i \N. separation Of maltir atiid ll~rir deost rhonldlliidcOI IltlltlltI)Ii'.phatces h% rcetwt-

plase htgh-pcrtilrinalcc 1Lt~Iltt clrintatligraphi\ N sIIIl IlIthttit aitpIl'attll. ti Lltlltlitatil ol

ttutht lated nTILClatltict III t)N N. 41 in lN /i1011a 119. 35-451 l182).

It [ I Iii m \-N. K. DII)( I RIN\I . N. .NNRi,. andt R %1N1i. Ii th iI i RS N I net'. anti rapid col, Irt It-

nc delerni inail))litn l I\t lchiiltne'.ira'. acti t it\ . Ito(' htem Ibh,oumt ,/ 7. 88-,)5 I 190 1 1.

Y I I,. I lItt R N. N. J. RE ,I iwi )I i i. N I I sitit. andi R. .1, R 5N)i ) I . Pro tin nea'ureniint ttith the

I,1111 phenol reagent. .1 l hi h(tii 193. 2W;-' 'S "l1 W I I

i ) NI 5'. I. k \'.1 \N N. NI 5N\. Ii CIi ( 1111. 1 1 KI 1)t\R, anti N. R s/iN. ell ktl-tpntn regtulationi

Elf etijkar'. tic IN \ nteth\ late lci.. hi.' J /iii(ihe 26(j. 8565 3-.X6 (ii'56

.1 )A B I ii I it. Preparing nuclei front ct-lit i nlioaerctue.'ith tartciiitg aitt tar liilliit-

ing sN nchriiuet cIIt-ltiiriiugh ihPelccc lial Bhi.ci' 141. 7()-' I l'154t

12 1' I- m\ R'Ki. D) J. Sit \ It ss-. and \I. I I uN-. Ipe-'iu \ ottSI lileeiini tuiittiiiii'In MOUN hautlt-

riihtatia nmetdiateti h.litti r\Icclcai tlse nniphi~i'haie. \anii, 233. 41 3-41 I 10-1 1

ISK N. F'it .stand N \t I Pa . %liirphiiltigI dtlereniTilil0 at ntiltite Ittriholattoiia telk l'. indet In

tb 6'. tiirti atienitine ;?' Sr,.lii. mtonoiiphtd e \ ii hi. 233. 14 t 142 (19 1 1II

)N-A MF I HYI AI)ION 1"97

?4 J. R, KA it s, R \Ni\ i i iNs.. and K. Si ill ISSINdi R. Induclion of acc sIchohnestcrase acti% it% in

mouse neuroblasioma tissue culture cells. .\ltur 229, 345-347 (1971 ).

35i Y. KIMtl1. C. PAi IR1 ). Sm C tOR, N. . xR %k. and (!. Z. lit x -I R, Maturation of neuroblastoniacells in the presence of dimeths Isulfoxide. Pr, , A( , Iced .S(i i S. 1 73, 46'2-466 1li'6).

.3 K N. PR \s*), X-ra\ -induced morphological dilcrentiation of mouse nturoblastoma cells in % itl o

\ aturc, 234. 471 -473 ( 197 I .I .-\M .No. E. Ri( iI i soN. and kf. NIRI Nfi R(;. Neurotransnitter s% nthcsis h\ nCUlO)h.ilOnia clones

Proc .All .lhad .5uel 1.1,I 69, 258-263 (1972).3,x N. I . 011 INI'K and R. C. Rt si ). Interrelationships betsecn ioniiing radiation, protein s\ nihc'is.

and the ph\ siological expressions of radiation damage. In tldi',' ut c, Rm,'t; h, 3 ,'ci (J. I I ell

and I. .dler. Eds.). pp. 107- 160. Academic Press. Ness York. 1976.3v R. \. (iJVRSI I and 1). W. MARTIN. Presence of a DNN demeth. lating actiit\ in the iw.cus of

murine erxthroleukemic cells. J Blt ('him 257, 8581-583i(1982)40) S. B. I.,oN and II. lI., DNA meth\,lation: The eni\mes and a naturalk occurring inhihitor. lcd

P'ro, 45, 19271 1986 ).

41 S. B. I )ON. L. Bt'i)Ni(X R i., and M. Mi it Ik. Naturall, occurring meth\.tation inhibitno I )N. hIpo-methlation and hemoglobin snthesis in human K562 cells. 1/,/ (cl/ IB, 7, I '5' I ,

42 N.J. SM.s, , 5. 1 Hot). F. J. Bi RN,. and S. J. (.xR ii. Activation ofc-m.c and c-K-ras oncogenes

in primar rat tuniots induced h ionizing radiation. ifi, (clif B,[ 7.932-915(198-)

F\p Brain RC, i9)) '5 5 E)(ei naBrain Research

,Springo.rA Ncrig 1989~

ARMED FORCES RAOOmLOGYRESEARCH4 IT1.fl

SCIENTIFIC AIM"t

SR89-20

Enhanced acoustic startle responding in ratswith radiation-induced hippocampal granule cell hypoplasia

GI. A. \licklc\ and J . L. Ferguson

Bch.i\II.t Scicncc DqI litli! Vm\ticd F~rccx R.Idil opI~u Rc~,rch lntfiiC. BCICtt~,. \11) 21151-5 14 _ I SA-

SummarN. I rradiat ion of tilhe neoanatlal rat h ippocami- are beinrg used i ncreasingi v in the study, of humnanpus recesic, the proli ferat ion (if granle1 Cells il the behavior (Wilkin-s et al . 1986) and thc helictoior ofdentate gs rus and result', in loconiotor h\ peractix it\ other animials ( Eaton !984). Although the acousticbehav ioral perseveration and deficits, onl suM' startle response is at relatively simnpie behax ior. itslearned tasks. In order to address the role of chanfL ensitiv it\ to at \ ariet\ of experimental treatments hasinl tiN6 ul u1.s salience: and hehax ioral In hibit ion illi made it an important tool in pharmnacolog\ andanimials 'A ith this type, of brain daae-irradiated toxicology (for reviewN see Eaton 1 984). In particular.and normal rats wecre compared in their startle brain mechatnismis of sensation, learning ( habitua-react ions to an alcoustic 1,timulu1.s. A port ion of thle tion, sensitization). miemnorN and miovemient arebrain ol W( rats mx as exposed to a fractionated total being elucidated through measures of startle (Davisdosc of 13 G,, during the first 16 dxspost partuin. 1984).This procedure produced seleetic h~ poplasiaf 91 ' ~ The neurons thal comprise the primry% acousticreduction)I of the granule cells in the hippocamipal startle circuit reside enitirels within the brain stemndentaite cyrus. Other rats ( = MI) were~ shamn (Davis 1984). 1lowr. the nature of' the extrinsicirradiated. Sudden tones, were presented to each neurail systemns that mnodlaite acoustic startle is not soadult rat at at rate of I e\xer\ 310 s (spaced triails) well understood. Since the hippocamnpus has, longduring an initial I (-min session and1er I c~r 15s been know n to play, a role in response inhibition

maizssed trials) during, a subsequen~tt ,cssloill. Irradi- (Douglas 1967: Kimble 1968: Altman et al . 1973) it isilted rajts startled with a consistentl\ higher amilplitude likel% that this Structure ilso influences stairtlethan controls anid wecre more likclx to exhlibit startle responding. In support of hippocampiillv mecdiatedresponse,,. These animals wkith hippocarrnpal dlamage behavioral inhibition other investigators hlavecalso failed to haitiuatc to the startl Ics til lLis, anMd. reported excitatory behlaviors alfter hippocampalunder certain circumstances, shlowedc poteitiated lesions. For exaimple. rats wvith hippocamnpal lesionsstartle responses after inlo. tone presentations. exhibit locomotor hy~peractivity (Ieitelbaium and

Key words: Startle - I lippocamrpus - D~entate i'x s" - Milner 1963: M-eans et Al. 1971 ),response persexeora-

(Inlcel- datn tion (Ilsaacson 1974). facilitated acquisition of activeyrai~l clls- Rdiaionavoidance ( lsaacson ct Ai. 1961 ) and impaired per-

formance on passix avoidance tasks, (BIlanchard andFial 1968: Isaacson aind Wickelgren 1902). More-selective hippocamnpal lesions of (A3i haels be

Introdctionreported to produce hxpcr-recictiit\ to sensory

'The startle response consists of at characteristic siuain(inerlrnad0tn18 .Sil h,cu,,:ic o r~pid111.1,cuar onracion hviningat function of the hippocam1puS In s tartle responding is

the l,)lthMid',C~j~C~lidl% io~oingt-h rick, controversial. Somec authorI (( iroves ct ail. 1974:the mi~uthandsequntill\i nolx ng he eck I aton 1981 ) havec reported that lesions of the

forclcgs, and tinrillo, the whole hod\ (Landis and 1 lUnt ipClrtlsontcnimtkaertrl,\Nhc1ipo3a9u do not,:il~n, consiste wtt lx ltr tirte wilI 99) -\alxsesd ox nl nt asocate wih tarle others, ( (ooer aind Lev\ine 1972) have found

increaised acsic_4C startle after sarilcally induced(Wltpit rtmr \ tki\.Ir~ c.h phippocampal damnage.

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Reiult% this dliffence-lC. 'I his fiact. inl itself. is not remarkablesrice others,, 13a1 er aitd \ltImri 1')751 hax e reported

)uir It oialind bhavtioral data~ sutrecst that different resuls from measuires Ot liippocamipalhippocampA~ -,riirrulC Cell h1\ pop1Laia1 enhanlce, sItrtle anatomy111 depenC~ding Onl thle planeI Of the b'lri sctionltilllIRItuC 1 ',irr rcICu\ irid limi1ts stale1 habil~ita- anle.The cerebeIllm was'. shielded duieirraidi-trIM. ation . the deitxII. Of the granule ,cIls present \\ia"

I \posure. ot the nleonaital rTl iippocirirpulis to nlorml anid tile orcn-mizatiori of eells xx thin tisl,rorii/im raiaitiorr produced at signiticatnt Itl) t 14.3. struLcture did rnot resemle~ that recpor-ted to occur1P I1111 depletion Ot deltalte -raiLeo cells hierr this brain r is ' exposed to on1lin1e- raidiationII able I . i.I ). I his darrItl-Cc xvs trillx quarifIhCdI Brurniir arnd Altman 1)74: Alrmarn PI . Further,.

orrllx ir tilt: birrs riidi rrrll selected for cell counirre- at redc(Itionl Ii er-cbellarr heiehit I ii tile' coronail plalnebut x'\ as ctsilx obscrx e~d i Al irradiated britirs. has, been obscrx ed Il ilrts, with raidliationl-inducedS1irm1lJI% . ho It 1i nIne areas aiid th I rlru 111 IIcCell cerebellar darieeg I Altniir et a)l. 11IS 1 hN ut I, as notLilsitics t t IIe IrrI ~ilrel dentat e er) cxxere sIi- preserit Ii ouri subjects. It riax be that re.ducedC,nirtlxl rcduICcd COiinp1IrCkl to those ill thne control rats cercbelar si/c inl thle salilit Ial plarnL'. but not tileN UHIj I 1 I i' III)1 aIrid It HI." I' P; lil(,(ill . coronal plattec. Is dfice to oxeralll cranlial shortenirre1-

rcspccrti\ l\ I I lie: spesltlcIt\ ot til', dirnnael-c is Jillus- sirnec exeri pmitial head rrrrindiatiori canI reduice b)oricIrtrard 1 thic 'p)m I it Ie 11isr-1nIti1c p rarirdil -,riomxli Iboth xx thlin aid outiIdc III ' lied) 1 Nioier

A I rnerniirns rhat xxeeL (lirCt l tilte p'ith tit thie \ arid larlsorns 11)7()).i~s Iri~ittiori piiduccd no chan-c Ii the tliickrne" Althione1li tine orcriit tine si11-iiel crellarikiirle ( At priill~i cell la'~cr. \ct thle tlnickrness Ii cliiee reported here is, current lx utneir-ca . cerebelli

the Lleiltre Lrmnu1le~. Lcll l i\cr- I sniriit iintI\ darinace~ pruibhil lli\ ed a rliiible rolc Ilr thei.reduocd Itl11 k) '1 I' illI. I lie -ritillk cll1 prodiltIOIir ot thne bchllix orall results lcpor'<, bCloxxp)Iipltucr~s 11 l... rnulinrllit II-ISLsII urirarea) ot thle I here is, a lissirniilirit betxcen thne bel~iiaxicr' nic-

IRRADIATED SHAM-IRRADIATED (CONTROL)

AC

CIO.

-1

d0(1

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D)iscussion

We,' report here at pocniaitioii of thle acoustic startleFe %kt lses In ratse I ad t ion'-indUCCCd ha ~iZlaia (of

*Illpocillpal dcnIUtC UraulIc Cells. Ti's obscrx atiollwkas madu during 2 test session,, (separated h\ 4 daxsin wkhich tones as crc prcsenited in cither at spaced ( Iper 30) s) or TmaSSCd ( I per 15 s) format. F liaionlof tile acoustic startlc response kis as not obsura cd inl

Hig. 2. \tCitn 'Iittptit. , Iwti 'tattle [c'pitt'cx eChhItCd 1'\ animlls With ippol)(campal~l damautiC.rr'Kidtic rit, kitfthi icrttipil eittll IfiUid The method of fractioned partial-brain x-irradia-

mird -i, Re'Lilt' lthI tell nijl 1'10k' LiCL hiimn it i tch k'

tci.te: c In i ' t los ii . '01[I-it prc'itwnN k el t' 't ion used 11crC has bcen shiow\n to produce at slcCtia c

%khilcU~f tnvin'Ii\%ecwic liclun U it ni" at , reduction In tile number of 1-ranIIC Culls, ill thu

,I IIllI Iudc' 1th" I ! 1 c , MtItr, k hfr"iti0 the 11UIittC IdIi heii I)Amnato 1 909: Baaker amid Altman 1 97~ Baea 'nard

11Wi lIt in rfit, i ih ltipp i~tii itniee1 tDi'.pcriirI MII 1f lIcters 1977). 1 owexcr. this damac in thc nIcoMIitll

iii. the 't,1ini)iRi Cirlirs 01 ftLt' top11 I*ILjrcsoilt .1 t tlstteiik hippocamnpus Imax also cauLsc sccondiiry anatomical'-~TItkii AW1!11 I Coite C te heC esjicriTiiCtett Ind C, ci titi -'1u chamizes. Zirmmler and his colleacs (IW85 1986)

it hrti time 1) pril. P' \t,int-\\ lititu\ t hat\ c shok\ ii that thc brain mla\ compensate for. thisCarl\ radiationl-indUCCd daIaIuC !o thle hippocanpall

urari nlc celIls by stimlulatine dc ndriti cur omi . Theirresults dernonstmi t that at rd cCIt ion Of aI sluci fic

I spitced trials) \\itas no t statisticallak si unlificanit for. nCuronal p)opulationl can induce: (I ) at comipcilsator\either experimental or corntrol animals M~ann-Whit- increase inl the 11curopil layecrs containing ticnca I,'). Hihc rcspoinses in the fi rst and second halacs dendrites of the remai ningi neurons. (2) at corie-of scssion 2' (maisscd trials) rca calcd at statisticalla spondingu relative increase in their aixonal pm cec-si ni ticant decline in the st artlcs cxh ibitcd ba the tions, and (3) at shift and expansion oif afferentski ii- i rridiiatd rats It* 67.7Th) -~ 2217. 1) =. i0 4 projections to an ad jaccnt neurIIOlit populaifon.H oss ck r . tor thle iradiated rats ks ith li po iiipal Thus. ah hough our hippocamnpal radiation producesdaiaLc . not on11I did startle amplitude falil to lhit ii- damnage specific to the LuranleI Cells, subsequentate d~urn ne tile second session but there w\as at trend brain charwes. in reaction to this initial darniac. milktossa rd poteniiat ion of thiis rusponse . prdue ore-pe rvasive changes in neCuroarnatolm

Bca use a rapid Ihbitt ion (if start Ic inl rats %ithi Although these datat reflect changes in h ippocampalhippocitmpil darnitiec has, beeni rcpoirted I Lcaton afferents and nt ra-hippocampal neuroanatomv\ . theI 98 I I aCiom pairision kis as made of tilc first aind last 3 mllos t dramatic atnd( direct raitd ioLe n ic damail ctrials (if the first 1)) trial block dur-11 ineech session. in observed inl (tur experiment can lie found withinl thlehc first session there as as at declinc of 2'in startle granleI cell layer oif the duntate _, crus.

amplitude of slirimidiaitd rats [Mann-Whitncv I' Some authors ( Groves et al, I1974: Leaton 1981111 .26) 3 495 . j < (05 and at decline of S(( for have previously reported thi;( lcsions of thle hip)-

irr-lididttcd ;;it, Inut Iiutc ntj tile sucond scssioni pocamnpus do iiot coinsistentlv alter startle, whlileI massed t ri ils) t here ast, as a5~2'1 declinei in startle otlhers I Coover aiid -cvi ne 1972) hake founddimiiplittldc: for shilrn-irradlIited ratls [ \lan-\hit - increased acoustic startle after this suricr\. Thenca t ' (.12) - 96. P' - ().Il I ad I ~ drop for lesion test interval may be at disuinguishiing featureirridlitcd ras jinot it a sieiiiicaimt tlrupl I butwecnl experinments that have found potentiatcd

Not Al toinc precuntations reulited fin sItirtfcs (ice. startle amplitudes and those studies iiot reportingmoinemnts that mect tilec critteria Cstiblishcd h\ Ouii these effects, in rats with liippocampal damiage.ipparailus suit imips). DI )rnt thli first session (s'paced Fnhanced startle has bcen fouind wkhen thcre askas,trils) tbu irradiited ralts tended to stiirilc nbore sienificant interaal betwueen the brain lesion aiid tilefremicitla thani thue control rats but this diffurunucc startle test. Ill both tile Currcnt experiment and that%\;s n olt staitistiealik sienlifiuanvt Duiring tic suCOnd (if Cookcr and LevinuC 119721 tile lesion test iiiter\ alscssit) iii mascd trils I thu higher frcqucncv of startle wais long Iapproximlately 194 aiid 7)) days. respec-

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AMMID FOOCI PACOWOLOGVA11111AAC INS#T1T11

8CIBUT1FIC REPOT

SR89-2 1Original Contribution

)0M F,)\I \ A A~ F.I. N I NI "IlF (IL I NLA PIG [-II PP(X Vv\ ri\L SI- KT,1

It i, C Pi I i \1 \i,, aml K m o\ I- Ni i

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11,1t '. anI~at d Il" artc t- ~ 1 l\Id ,I(tN I lm i il Call all ndi I u te aia' r dtecu. thc:'11" cl0 iltltc ,,'' Il lai b 17' Ik ti~- ill IllIN fill thelcLI t till:,ti h i~d n\ hoa ticI ad -h

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it A '-thf .,,.Npcr ie I .ourccIpetho c Iicltiit,it r0 Pecihd Ih lc\11))4) r hik cc4

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l~cll a' othicr alcrct to thc CA-\I 11 ranIULIal kCclt'. l

idd t i ~iI l/ 1red intaI ocr rcottd 'rc0 0.4 0.8 1.2

c~a' iiicrlclctridc tiic~ ~ ih l NCI nd na~IncVoe\ (mV)

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pIiOPlatilini1 Npikc 11 thc e rcflial r-cciidcd ation >0r 0.6pitctCialJ C:LrIl i\JTOI ~ Inl a poplakltin 0I('.A I pr rmannl ccli' In) Nomer cpcinlicnit'. a 'cconId...........

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F-ollovO T' lt~ccnicnt ofi thc cIcctriidcN. ha'cic i-c-SI> 3ciircditic %kcrc itaincd for a ni in 011C it -M nun to EcIn'ur-c Ntahilit\ ol thc tP, ,uc. StnIIuLJLI ~tcIn'it\ 1'Nct to it \jaluc that prodUccd 11pl'o\lnnltcl\ a h~alt-Irrax- 2MIA rc'pinc . It clinc11- til, pcriod tlic IrCponl'C' Wl

chancced 'NLhstantIIr . the cLprmict L ' 'cardcd.0

0-0 0.2 0.4 0.6 0.8

PSP Slope (mV/msec)

Iinucd line .\ Giraphli 1M t~ L~~ hiloranc- I vckltic Iic pl-k

/ [lioi pike iir - .,iiiclrcili willcr diii1TIL11C BI ( 11,lilliic

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Life Sciences, Vol. 44, pp. 1185-1191 Pergamon Press

Printed in the U.S.A.

A $NWE PO M S NI A01OO LO YV

0IaNTIFIC REPOT

SR89-22

RADIATION-INDUCED VOLATILE HYDROCARBON PRODUCTION

IN PLATELETS

E. Radha*, Y. N. Vaishnav, K. S. Kumar, and J. F. Weiss

Radiation Biochemistry Department, Armed Forces Radiobiology ResearchInstitute, Bethesda, MD 20814-5145

(Received in final form February 20, 1989)

Summary

G .tLration of volatile hydrocarbons (ethane, pentane) as ameasure of lipid peroxidation was followed in preparations fromplatelet-rich plasma irradiated in vitro. The hydrocarbons in theheadspace of sealed vials containing irradiated and nonirrac ,.ted washedplatelets, platelet-rich plasma, or platelet-poor plasma increased withtime. The major hydrocarbon, pentane, increased linearly andsignificantly with increasing log radiation dose, suggesting that reactiveoxygen species induced by ionizing radiation result in lipid peroxidation.Measurements of lipid peroxidation products may give an indication ofsuboptimal quality of stored and/or irradiated platelets.

Thrombocytopenia plays an important role in the development of thepostirradiation hemorrhagic syndrome (1). Although destruction of plateletprecursors in bone marrow is a major effect of high-dose radiation exposure, theeffects of radiation on preformed platelets are unclear. The latter is also of concernwith respect to blood-banking practices since platelets are often irradiated at dosesin the range of 20-50 Gy before transfusions to prevent graft-versus-host disease.With increasing emphasis on allogenic and autologous bone marrow transplantation,transfusions of irzadiated platelets are likely to rise. Transfusions of platelets werecritical in the treatment of Chernobyl radiation-accident victims (2).

The effect of in vitro irradiation of platelets has been the subject of a numberof studies, but there is no consensus on the subject. The survival of plateletsexposed to radiation doses up to 750 Gy before transfusion remained unchanged(3,4). Another study, however, indicated that irradiation with 50 Gy beforetransfusion to normal subjects resulted in decreased platelet recovery, althoughplatelet function did not appear to be affected (5). In most in vitro studies ofirradiated platelets, no changes in the structural, functional, and metaboic propertiesof platelets were observed even at high radiation doses (6-8). However, irradiation ofexperimental animals appears to alter the function of surviving platelets (1,9).

Radiation injury is most often attributed to cellular DNA damage, and sinceplatelets do not have nuclei they are good models for studying the effects ofradiation on the functional characteristics of membranes. Free radicals, especially thehydroxyl radical generated during radiation exposure, can promote membrane lipidperoxidation (10) and alter the properties of cell membranes. Irradiation of platelets(100 Gy and greater) under conditions promoting hydroxyl radical or hydrogen

*Present address: DCLD, Food and Drug Administration, 8757 Georgia

Ave., Silver Spring, MD 20910

0024-3205/89 $3.00 + .00Copyright (c) 1989 Pergamon Press plc

1186 Lipid Peroxidation in Platelets Vol. 44, No. 17, 1989

peroxide formation resulted in inhibition of ADP-induced platelet aggregation (11).Whole-body irradiation has been shown to alter the lipid composition andarachidonic acid metabolism in rat platelets (9). Lipid peroxidation is morepronounced in old than in young circulating platelets, and it has been suggestedthat this may be a cause of platelet aging (12).

In the present study, we quantified radiation-induced lipid peroxidation in vitroby the evolution of volatile hydrocarbons, a method we used previously todemonstrate radiation-induced lipid peroxidation in microsomes (13).

Methods

Platelet-rich plasma (PRP) was prepared from whole-blood donations bycentrifuging at 100 g for 20 min. PRP thus separated was irradiated at 50, 100,200, and 400 Gy (10 Gy/min) using a cobalt-60 source. Platelet counts were donein control and irradiated samples using a Coulter counter. Hydrocarbon gencrationwas measured in PRP, platelet-poor plasma (PPP), and washed platelets. Theirradiated PRP was split into subsamples to prepare PPP and washed platelets.Washed platelets were resuspended in Hank's buffer, pH 7.4. Cell counts wereadjusted to 109/4 ml in PRP and washed platelets. Four-ml aliquots of PRP, PPP,and washed platelets were taken in 10 ml siliconized glass vials, which were sealedwith Teflon-coated butyl rubber septa with aluminum caps and star springs (Perkin-Elmer, Rockville, MD). Gas in the headspace of the vials was sampled with a gas-tight syringe for analysis of volatile hydrocarbons by gas chromatography using aPerkin-Elmer Sigma 3B FID chromatograph with Sigma 15 data system. Analyseswere done isothermally (60 0 C) with a stainless steel column (3.2 mm x 2 m)containing Poresil B (80/100 mesh, Applied Science Labs, State College, PA).Hydrocarbon standards (approx. 100 ppm in helium) were obtained from ScottSpecialty Gases, Plumsteadville, PA, and scientific grade air, helium, and hydrogenwere purchased from MG Industries, Valley Forge, PA. The first samples (zerotime) were analyzed within 30 min after radiation exposures were terminated, andthe same preparations were analyzed at different time points.

Results and Discussion

Pentane evolution as a function of radiation dose and time after irradiation inPRP, PPP, and washed platelets is shown in Figs. 1, 2, and 3, respectively. Asignificant increase in lipid peroxidation as measured by pentaue evolution wasobserved in all fractions after 50-Gy exposure. The increase in pentane evolutionappears linear at lower radiation doses and earlier time periods. The relation topentane evolution and radiation dose is best described by a logarithmic plot; Fig. 4shows pentane evolution in the three fractions at the 6-hr time period. Extensionof the lines to a point where there would be no detectable effect suggests that therewould be no significant increase in pentane production at radiation doses less than25 Gy. The slope of pentane evolution with respect to log dose was different forwashed platelets than for PPP and PRP.

In general, ethane evolution followed the same pattern as pentane evolution, butthe ethane concentrations were much lower. Evolution of the two hydrocarbons wasnot strictly correlated, e.g., the slope of pentane concentration with log radiationdose was steeper compared to the slope of ethane evolution (Fig. 5). Similarly theslope of pentane production versus time was steeper (data not shown).

The spontaneous release of pentane over time in platelets (controls, Figs. 1-3)indicates that lipid peroxidation occurs in stored platelets. In vitro irradiation ofp!atelets induces lipid peroxidation in a dose- and time-dependent manner. If it can

Vol. 44, No. 17, 1989 Lipid Peroxidation in Platelets 1187

16 __CONTROL

-50 By

100 BY

(1 12- 200 By

0- [ 400 Sy

zE J

z

Z 8-w_

0I0U

a4-

r f

0.0 HR 3 HR 6 HR 24 HR

POSTlRRADIATIQN TIME (HR')

Fig. 1. Radiation-induced pentane generation in platelet-rich plasma (PRP).

Values are means of 6 determinations ± SEM.

16 C CONTROL

50 6/

too ry

Tr' 2oo ry

a 12 4oo sa- 400 By

-J

[i

z

zB

rk4-j0 -

0

aL 41

0 HR 3 HR 6 HR 24 HR

POSTIRRADIATION TIME (HR)

Fig. 2. Radiation-induced pentane generation in platelet-poor plasma (PPP)Values are means of 6 determinations ± SEM.

1188 Lipid Peroxidation in Platelets Vol. 44, No. 17, 1989

61EJCONTROL

!50 By

1 ~400 G)y

-j 4-

wzF_

zU'jIa-j0702-U

0.0 HR 3 HR 6 HR 24 HR

POSTIRRADIATION TIME (HR)

Fig. 3. Radiation-induced pentane generation in washed platelets (WP). Valuesare means of 6 determinations ±t SEM.

12 ' set-Rlch Plasmam Ptatelet-Poor Plasma

10- A Washed Platelets

6-

4"A

cc A

0 1 iI I I INonirradlated 50 75100 150 200 300 400

Log Radlation Dose (Gy)

Fig. 4. Comparison of pentane generation from PRP, PPP, and WP at 6 hrafter irradiation. Values are means of 8 determinations.

ol. . , 1 ) lipid Pcroxilition in PI1 tLI ts 1189

10-

A Ethane

8 Pentane

6pmoi

Pentane/mI4

A

000 I ,I II I I

Nonirradiated 50 75 100 150 200 300 400

Log Radiation Dose (Gy)

Fig. 5. Comparison of ethane and pntane generation in PRP at 6 hr afterirradiation. Values are means of 6 determinations.

be shown definitively that lipid peroxidation is related to detrimental effects inplatelets, then precautionary measures can be taken with respect to storage andirradiation procedures in blood banks. The dose of radiation used in this study, atwhich there was an increase in lipid peroxidation, was comparable to that used inremoving lymphocytes in the preparation of PRP. It is interesting to note thatthere was less lipid peroxidation in washed platelets than in PRP. One possibleexplanation is that platelets contain efficient defense mechanisms against oxidativedamage. It appears, for example, that platelets have the inherent capacity toregenerate glutathione in vitro, at least for 48 hr, but total glutathione decreasesupon storage (14). PPP showed the largest amount of radiation-induced lipidperoxidation.

The major hydrocarbon formed, pentane, is derived from the oxidatiun ofomega-6 fatty acids, predominantly arachidonic acid. The minor hydrocarbonformed, ethane, is derived from ornega-3 fatty acids, mainly linoleic acid. Pentaneproduction may be related not only to nonspecific membrane lipid peroxidation butalso to the high degree of oxidative arachidonic acid metabolism involved in theformation of prostaglandins and leukotrienes in platelets. The relatively nonspecificindicator of lipid peroxidation, malondialdehyde, has been used as an indicator ofprostaglandin endoperoxide formation by the platelets, occurring during releasereactions (15). Malondialdehyde formation has also been considered a sensitiveindicator of platelet hyperaggregatability seen in certain disease states such asdiabetes (16). The exact relationship between pentane formation and "norminal"oxidative metabolism of arachidonic acid in relation to platelet function, as opposedto "uncontrolled" peroxidation of arachidonic acid, requires further investigation.

The relationship between the observed radiation-induced lipid peroxidation andchanges in platelet function postirradiation is also not clear. We have shownhyperaggregatability of platelets irradiated in vitro. There is also some indicationfrom in vivo studies that their function (platelet aggregation) is increased shortly

1190 Lipid Peroxid.ition in Plitelers Vol. 44, No. 17, 1989

aftet irradiation, before platelet numbers decrease (1). Platelet activation is oftenassociated with an increase in cytosolic free calcium concentration, partly due tocalcium release from the dense tubular system (17). We have also found an increasein intracellular calcium concentration in irradiated plat-lets (18). Peroxidation ofmembrane lipids is often acccompanied by structural and functional changesresulting in impairment of ion homeostasis leading to Ca 2 + overlcad and cell death.In the case of resistant cells, such as platelets, cell death does not occur readily,and gross changes are difficult to observe after oxidative damage, such as radhi.,-'exposure. Our results do suggest that radiation-induced lipid peroxidation mayresult in alteration of calcium homeostasis that is reflected in observable functionalalterations. Further studies are needed to understand the exact nature of lipidperoxidation involvement in platelet aggregation. From a practical point of view.the effect of the oxidative changes due to aging, radiation, and other storageconditions is relevant to platelet therapy.

Acknowledgements

This research was supported by the Armed Forces Radiobiology ResearchInstitute, Defense Nuclear Agency (DNA), under work unit 00162. Views presentedin this paper are those of the authors; no endorsement by DNA has been given orshould be inferred. Dr. Radha was a National Research Council research associate atthe Aried Forces Radiobiology Research Institute The assistance of William E.Jackson III for statistical analysis is gratefully acknowledged.

References

1. T. P. U. WUSTROW, H. M. FRITSCHE and 0. MESSERSCHMIDT, ThePathophysiology of Combined Injury and Trauma (R. I. WALKER, D. F.GRUBER, T. J. MacVITTIE and J. J. CONKLIN, eds.), pp. 125-135.University Park Press, Baltimore (1985).

2. R. W. YOUNG, Pharmac. Ther. 39 27-32 (1988).3. M. L. GREENBERG, A. D. CHANANA, E. P. CRONKITE, L. M.

SCHIFFER and P. A. STRYCKMANS, Radiat. Res. 35 147-154 (1968).4. L. N. BUTTON, W. C. DeWOLF, P. E. NEWBURGER, M. S. JACOBSON

and S. V. KEVY, Transfusion 21 419-426 (1981).5. A. E. KALOVIDOURIS and A. CG. PAPAYANNIS, Acta Radiol. [Oncol.1 20

333-336 (1981).G. M. J. STUART, Transfusion 23 106-108 (1983).7. M. V. PILLAI, G. D. QURESHI, R. HOWELLS, N. NARASIMHACHARI, E.

G. BEHM and T. A. HAZRA, Milit. Med. 150 496-498 (1985).8. G. MOROFF, V. M. GEORGE, A. M. SIEGL and N. L. C. LUBAN,

Transfusion 26 453-456 (1986).9. J. L. LOGNONNE, R. DuCOUSSO, G. ROCQUET and J. F. KERGONOU,

Biochimie 67 1015-1021 (1985).10. J. C. EDWARDS, D. CHAPMAN, W. A. CRAMP and M. B. YATVIN, Prog.

Biophys. Mol. Biol. 43 71-93 (1985).11. N. 1. KRINSKY, D. G. SLADDIN and P. H. IEVINE, Oxygen and Ox -

Radicals in Chemistry and Biology (M. A. J. Rodgers and E. L. Powers, eds.),pp. 153-160, Academic Press, New York (1981).

12. M. OKUMA, M. STEINER and M. BALDINI, Blood 34 712-716 (1969).13. C. R. DOBBS, K. S. KUMAR, J. F. WEISS and G. -N. CATRAVAS, Int. J.

Radiat. Biol. 39 445-449 (1981).14. E. RADHA, T. D. HILL, G. H. R. RAO and J. G. WHITE, Thromb. Res.

40 823-831 (1985).15. J. B. SMITH, C. M. INGERMAN and M. J. SILVER, J. Lab. Clin. Med. 88

167-172 (1976).16. M. J. STUART, Thromb. Haemost. 42 649-65 (1979).

71FUWI T HREDF FO2*SADIOM ~OOGY RESDRWUMN iii7~ 1REPORTS APRIL-JUNE 1989(U) ARMED FORCES RADIOBIOLOGY

JUNLRSSIFIED RESEARCH INST BETHESDA RD JUL FI897 N

USI FIDti /Emmhhhh hil

''III111 6 I136

I~r s1l li.t I I1 I.'' sT ia IIII. 1 t y x))~rl I I (ILH emlatolo)gy

ARMED FORCES RADOSOIOLOGYREFS E AR0CH M~STrTU TE

Long-termi Culture of Canine Bone MNarrowii Cells SIETIICREOR

k hstract I ) ri t I , T I' i I 11 l ia ~ I I i I t I, t i ite, m it Il c I,~ I [cii1 kcnt ia II I s .1 pptIl aili 11 l 1. A x 'nt IIh sIIIII ail\cc i'~ T I IC-t 11%t 1Tii 1r\\ c,1T T 1i1ii arc cilccI :lel. I Ilc cili'l t\ C III c ItILI Ti 1\ Itllihllita cclis , IhI t anlin lclniruis

A ! " ( 1 iii 1 1 1 al l 1 , 1 I t I nc 1It Il l 11 C I5 i t LC A l L i l ' \i t ic ci l I ~', c 0 1~l t i L Ii ! i u I I l ) i n [IS l ' II .l I n I C t i i -I f ( ) kI i " I \ T i l p h ( t r i '

1v its 1 [I 11 L' III n A th1 nr~Chent ,cHlI I Iact InI I S1inig this [ s]. S cl.c I\L' arei I cit rCntis sCalirchi 1(11r [1tctbids to 11i-t Illt'r t h lII: It L t1 I Ill hccr if ( I I ( 1 \\,I', 0hibtii JI) IL il Cf c\ tit ICIT(IS M~il 12CtIC' tra,11fcr Into) cannulC

.5 CT. !hal' \ -'II'N% IMc LiIIc'CLI At Il R i JU11- 164() lhCrI()(IClic pricicolIr~ cci'l cultUred till "4 11 \sitlh tI',I, , 1%K ' % Mldil :M \% lupieenec Wls I)[ ''rcrnd pocuICIMc paickactiing Ceills I II ( 'Ill 1)Io'SIbie Mrethold could

1 tini %\ I th cit iflitii ll 'I)\ilIi',ll (I: -( I N1)i bL'II to l InCrsCa the itiMe0that niII s I cIl\ikel', .11C C\p)115'cl t0

'm1ilss. Il b11 e hi.cclI \ 1)iS it 0Iti the flhiti,1kilict-C'lt Cell let]()\ Hial \ CLIrOs h\ C011t1711111 ng 24-h1 LcIll\ti attln Oil [)ic'k-lial ITI in 2"-S I Ises :itcr fchareine: theI eUitarcs \sIth0 achitigcCli's Ith1 loictV-icrot1 T1iarross' Ic uiturcI Id \%caclsttll

I ttIIlIn11l1 lei J iI] tlo )i L ' II 11111Tt I Cll', I ,\ccl, after H it IS-CiOtaininIC1 supernatant.e',hisiethc stVit,,i- cell ;,I\-I Inl tisl' stud\ \%C diCs,crh I nictto orlCUAI * tbchoi-e cL I-

ILIIc 01' itliitc" Madrls 0\Cel, bIsc14d On nrodfction101S o1'flei- 4) 17' 7- '1! il. tl~ II J!I ; - U T I II n l lITc S 1 c

llnetc-nt one irissc~lItcirc5 (otcr :it In I. iro( approtach MIaterials and methodsI I I ifl In t1e!eIC I IA t Irts IIL 1 1 n c a itnI s lcoti tII II nug 1e l c o po I Ct I I. . ;)701111uii a 00111 A l W (1 1 atn' ot bolll~ 01 th ,L'\L-s ,nud \ ur-

Ill &es elIncItI.1. I hIs nlctItOcl %\, (KtsI clc'',ctIbCd loIVt Log- ;,\i%,bree', ibeaus'. to\ homund', and mniwel'u. I-i 4 nronthi of

1 TIll ,i eidlir (d Mi)' cis c niarros cll' 11j. \k loc I prodUecci age. C xxr ld as1 iniro donorsi titcs escperiientis. A\ninialsIi lllc j1':1l(I\ -h It tiic and g' CIIC eratrtioc J-\ci ihc as 1 tbc' I rccl fil--i:r'n or'r Rescareb( h t

51 ~ ~ ~ ~ ~ ~ ~ 11 j)t Uts -In, f nrhsCd Irim coil! nICTrCial t Iliit Statces Decpartnmcnt oit \gri-niacrI phiwc III''s -1( till mvI Isht' \( Fl -( iN I - peruicds of' -ulIir-CnsCII cilacr. I )ous I\cre dluaraittinti l on arri\ al scree'ned

Il ni1it 10r 1C. dcjlclhc'tILti IttI!!'iits strain1 [1. HCmcr1- ti ()I de\uRCeIiL ofisc'i. and tcoilIinnd llr amiinium of1.11012 monthspmle', ill tis Sslcl stet 5,1 clclfccIV It&I tipotII thc' ptVcscnCC o)I bciorc' being icic~'cd lfor- [Isc. -NIf dud', "cre cc's'oiied and \accI-

,IlJcclVII11C stiMial iclls cotslstiig ilalc illr rabiecs. cfistcrmpcr. ltoprs.hctti adpars o\ rus.,iii ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ie acleeh vsc l iars cciI cc grcoup housec in an Arci Association lor Accrchd-

a htlciIc~eic'tispoptlatlin01 cii V pcs. ticudin phgo- tiin of Lahoratoir\ Animal ('arce IAAALAUl-accrcdited Ihciiit\ intcitlo)Il TIlCilca teli,,. e-ncioilial clils. and wnant lipid- standard indoor runs,. and proovidcd commecrciai dog chow% and chin-

lacli aid I pit c tes [ .rinatcd tan ss ate, cod libitumn Anirniai hoiding arceas wverc' maintaincedIli alliioi)to repl t onl muLritIc' iinct-term, aroscul- at

7iA' - 21[ %iih 50"o. i- 10 eiaic hunit using at least 15tiles.sit ilr Ino-elmecituie',birSs an ian str [1. ro- air changc% per hour of 100"r, conditioned fresh air. The dogs IIc'rel-I-L'_I oL~~~ ' \Ilnlaitr[1 r- (n a I 2-h light clark f'uil spectrumn lighting ecice xx ith no twilihght.

sinitin / ',a!,i 4th Itree: shrc',% 1 [4i. andic hunman niarrooN [5111I' C cil clescI rlibecl So Ilr. thic'rc' has bctii no rcport oin long- /1' 'l2C 1)1i10l11 Jafll~o,1i All cliigs "xere anestheticcl li\ is\. injectionILtcr CIiltnrc's i llicL bioic niarris. cells. \\e csscrc intet- of* ietamx Ii oar-'xet) at iiAl13 Ing kg hocd\ "ecight and the col-est cc t il estlilishl Iict t~c lwtil log-tr culture of' CLctill site cisepticalh ceansedc " tillt apmos irinc scrub and atlcohl

- ~~~ritiseBon lliiiniarrlox \\as obtained fronti thce huniral Cc' le x ialaiiuiici nirr' lcll', till 'r 15 rc'asiins: irst. miaintaining mazr- v.,cdl. aspiration uISIng a sterile 2i1-ml%,,i ingcandL 14-gauge dispos-

rl Cx ~ ll5 t lorici-tcrtit cLII ciltcir 'I iuhbe Uscec as a meanls of' able nILccll. fel l 1 bone111 nularross ikerc coillectcec front each cdogdepleting nmalicnti cells rim tic martlics - as has becen sug- it -ocA intcix ci,

Lieted b\ studc~ies % ,t1h ItTiritIIC'l nilItllblastic411 ICu~kenita [hI 1,11Ii,mc "I /'eiiowljn Ii iiuiirc Moinonuccle'ar marrotNJ~ld It) jl~illt'llls %oith cells nIi~ihhah Nihrni N(\ 10 )i IC x cl tUrecd in '-emcr canted-neck flasks

it mriinui. orning. NcIs' Noirkl i 2 -r0 %iI nil in 10 i RPl\Il-I I4i I ilttciin (MN. A. I3iuproduLctx. \cikers,,ilIe. Marx land) suipple-itieiitlx ith 2III preciercnc'd heat - inact i \ itc'd hiirs -scrum I (fll nio.

pt,,, iTic ill T1is pi.tlei tilet ' Ill "C(th -lti~'.Tl ii~~-2 1 1 1 . 11115 I lo . ibia iiiiic-. RiCks lld'. larlandl). Ill \1N hx-!!rIT [), 01. l ie II~ t) i s \ ih d leo IL e lc C l,'cti Vi\CTi Ill shiouilcd bn

-Illsc Ill) llrtiMITIC-2 I -Phpltsitac '11911mJ. St I Mii,.N issouri). il onl-Iii RscIT.ji sIWIltic d cc Licdltg to thpni plecon cs',eital amnino acids. 14'- p~ riiate. '21 i -glitainiinc. aincd P,, pent-

; It ill ill ( wile I'l lie ( tit i d I St. (dl I a,illior% \niials cilliit-siteptomxcin In soinc ecrinicrts RPI' I-I 41)niecliun xoaslellielhi T, IisiiaicIl IIllr~ic \nnia Resures II(li tcpl~icdc I.%th ceithc-r ilpha mninrun esscitilal iitcdiumi (M. A. Itio-\ii Til \ill~ii I c Ti'l \tlllia Rccar IiI ouiiulprlldliic'll. M~C( ox, 'SA\ riic'dtimII I(i1 1 () (li land [Id Ness York),1:1a [hI~ I ( I SIlilC1tlig. I red llutcliinsin or IFuschc'r's nieclicimT ((till( 'iI ( Ultures xsce maintained at 37"(

iniR,,-i h Inlt. 1 1141 l .injibilj Istreet. Sec'ttlc. kA -\ 98 1ii4, inl a ltntililiecl itmispllee of'S"ll (I ( in air. .Aft'r I %%eek oltculture.I 111111 I(IIad ii crent cells an ninic'l nc nir crc rnoo15Cc fromt the flasks anrd

kF( xII( Ixt iI I iv fIa F'Iii,iilgk k ()I I 'lti

F LiI I lxk 2I Lixk I Iaxk 4 F dx

* 1 1 111'x r , it -i \ 1 11 1 I litI I it I -(NI ci l I

N''~ ~~~ 2' )NN N4 i41IHi

V IN' 1 h' c kLx ' IN it ' it atI i'ii, 1'11LI'k h 1 \ 1 11111 'Nh II \ 1 tx Lim i

I..... A !tI I I' I I I I i' 1 2 4>'lp ll m l ' \ IIdif 111 ( h i 2 sIIII

!II, 1 2 !rj 2 I' ,, 1ll IN I. I 4't IN I, Na1)11r I h

, 1 1 x n' C x I , ii 1 ' it l il I, h I'l I 111M I'llx i II "Illx I xxi - i d - m adxx iili

\. 1~ 1 I ii. hx . 1 -Il cxI , IIJ ~j: 'Il 11,1 \ t lxi di t I llI ti lt'i lieo I[tI

Ixll.xri Ixx Ifnx~l tie Int nit(It",xrcIi I xI'I I 1 Ik0x49( 1UiII 1ix11 1 1& N a l I Ii h IIkl iui.1IINII(r 41

I t i t! I, iii Inrirnx Iin ihlii ixi xL!i dI I'xc i~u Iii (TN't" ItI -(i\ 1*~ Il \ (IL L fil-l

IfI Bk 'itiexi ix Diixn l il ". I 2tm \IN( in'' wall Iii andi~ d41' i

It I l c bI I ' jIII, I ICII I in i-' tlixr'l l iiiixiliie I dB 1 )t 194 x

Mul dl l Ilikx ut x ii ee l iti irxcI h x li iii m xxiiI INS. I'iii iii I %IN4 to aiii 2S0 1(II \

I xi !, I N II, ,i 11i ' 1 I I i tr I c I lnxii [ I i iuiiii jixin, ) Ixi c IIN Ix 4lx x I, II t\ tulli 11Itx irhixi F KIf Nl 'N m xiiii \aI(II L I I) R PM I2 x 94

4, 1 Ii li nt I 1 !IN ,-, 111 1,, l i It hot OCT liixin Mi I)N t 11x~it 1526 1xi

Lx 11 l jcnxi Ixrl liirI I'l N imxil)It' x Ixi'' I If iiur Lxti- cxndiixxnc I -e N FIo 't e 10 ID C-IIC l' 1,% l ,tl ~ i f h

[)I 'et .' xix NIx'Ixix I l Ie lxxiii llitex11 il'' 1 )I iii it (I Iri Ii I t (t ii c x n -trm linarr % I C11xx,1-', \ cil er (]titltehung ait xThe i teoll

ixti tl L rjei L d iiIiii )ix Inii tI Ic 1Iiirxx cltixIq Lher s nith5. r larr \I ( a xi nitinaltnin Iani il t r coInIitions

,iii tI 10xtiei r clx ii ilnxtil\xccts

1~~~~~~~~~~~~~~~~~~oti ,i irxtit inlxelr ilxtli ulr.ar iiiitl ~ esm ila Flit ittdiereu cel( ls -as xla lit sums oF FdheI~~~cl trcto liihii iltxx txhreti icinsixi L'xir Iiic ai findin thatutrhattm

ix lb ' F~cJ N2 ertihddiiix lucill lxiii ct tixicil tu Ni.-i tOIlli% Its e ixng-Irminiarrtantuluros txre t ote similr. ibisxhert-

xc> Plitxxirtliiti lxxix iIi )1 ii i- eceixin til rixtip xlii-the sa m parinosts aspralo t I Flu' sante cu1illt', odiliornsiL N'' kN cixon iiar. quantiIsL ohf (lk -( iN!nier onadhT !ren1

Ilmll~ ~ ~ ~ ~ ~ ~ ~ ~ ~~cl titFra1Tok'~l-loni h ae utr LIc Lnio ding tie sam CHur -ittle aube Findn hehak M1su1MLts oIt'nlT(MLCII rlr been contcrh nun rhrgigne the depe1-cndol escol he m o sdta nrot

hcr, (dII(rI;Ih~f-t1 LL11, n te fa,,s k~cic imiara d Mii ni ahoth (ioipriltg te hl Clriisx Fipo Iitii as qualife o i blen

cr 0C kCI I-A 1orl Coltall~d 111lar niont ulo urepr cxdilorn ecuste oneiion, nualwr on ( FI '-( M

FI I -( N%1 ( )net steek aFlt-r Lt hub iitg an adhitrtent ctell n mltI)'r- in Ft' rionadlitrent ttelF fraiction duriing the culture10la t il t . Hlx li itliur lasiks, Ixtreet L- ar1g' x i'll)h tle Samie tilnit.notititber (of winrtitx L- ttlls rxi Ft' sarlilt iiarristo aspiraion.\t xxxel' Is intlrk als. cells iinondhetl l to etic llurt: flasks om arw ifhilio l i xdb/hxtill iidtir o tfio

xxxLrC i (IxiItiel indtl Itled in) ( I I '-(IN! asasI able I shostslilt, xk l (totitls )I Ft nxiatllimrit tt'lk t tac. lask and We studied Ft' n uec 11LI i)C 0 iir' -111- l'rvne~-Iitire on ( I

it'C ii iiii her iF FI N(It I- 1, x 'dit ii kl r lion. viM number in long-term marrowiI CLlur b\ in-uihating a

I ,I I Ic Ic IrII i rI I mi-k II)I ru i %I'lr~l I iilitie41

R '1,1 ,(1I' L I I I I4., Iieiir oI~ I\ ircniNiplia riredruni Fischer's medium

\oni'f ihc- Nilidd- Niiiiad-hcvc c-r I licrcnit herent

ii I- cc'Celli cellskc ' f - lI' (I (lI' IF ( 1I I)I (- (N

1 l5f2 (4)114 n, 34 201 18I9,1S - 211 14 .. ')'), 17 1(N

1241-1 i;-4 5 14344 14 4- io 12u 1 143

4W2 41- 12.iilr. 13-

I bl . '11) ['X I"- ~ cIt III I' I I C~T 0,L)..)1~ OTi i f Icirtli m iicc Im iti III 4ij r~ ,I \ lt\( cr ilii 16)1 ( t -

~N1 'I A 'V 4 "M1N- 1~Vc~l~ic)icrisjiirti-.niic 1fikicii, )idriijLtlsiint: h\dricortisone

\cuacf .,1- Nimnurd- Ncinad- Nomnad-III111 6.-rent herci-it hecrent hereni

".11- cc.1-k ccfic clk- cell

I-t -T' 2 -is 10( M 44.2()1 1 1 (Wi Ini 8i3)ii2 o W>. I6 120T 1 33o -. 'k~

24 1,1 41).)" ii 14 1) 245 A it-I 1- 1i 24 I. 114 1 IT ij.(14( Is I S s

12' 4 1 ) 3 - "S 31041 6 Ssf1 I 21

4 i 111 4i -. "W I I50k 14'1(1-,) I25 5 14 1 ffN I I,; S

LI .. !.:- 1c- c l I L!,-'I I 'ec A 141 c-.tl I IiY ng anl adIliecri t -el I fa\ cr %% I t 4.5 I lI %IN( V It ai ir'ing 2571( F- -.40IH

,lIILIIC 11,1',k it fici 13 i ( . Both flasks kcere rciarged tic esperirierits. table I 'hIM%, the reCsUlts 0f one ofthe fi\eailici i \eck %%t OTC1 heaml nInMiCIr of riarrisc cclls 013 - C'\p 11iflelits.I I \l \( , ( .ntalinilil IS' 13 ( H -C \ I fun) the, sale niar- N\t.I \ C C511) a lollir-terni Iltr 1111 C %nh R PMI1- 1640) me-I,.% t,,apmrtuIn i \t \lccklc fietrs al. cells, ilofadhecrent to the d111um1 sulemente1CIId %kciii 20', prsCreenedC kIal Call" Serum

ast k' (ocr 'i ited an rtPlated i C TI, -(Il assac s. Table 2 instead ot'horse seru ni l. The flask ccdk as rechareed a her I \CC eksh- Ii \%e cueckic ciLlfts ol' nrinadlierett cell in ach flask \% CO111MI I Ii MIN .. Ioiarr 3111C F - N! h

,1nd Ih 11C i~x !)! ( Il -( It \1ile 1ii'nadherentm cell traction- culture %%as niaintained f'or S,- sceekl. VhIl number of' \INI lic -.umi Ot the \%eckl\ (Fl -(,\I couts1' hcsIighest tOr thle InI the ioriad~herent cell fractrion rapidl\ declined. There %%as

ila-.Ik nT.IcIitCd at 1-( 1 he sUper-inrit oii 3-( has, been nIt( ( F1 -( I \ fross III durlig the cutr perrCJIT od ot' I% %\ eeLc'riiiInicd! In tc\iiio pnetep~net dt not shoss il. %% hen PlatI ig the nlonadlierT c~ll in aT I I -( Il %Is . 11 hrs

fin ding ~ has be crilf C filled r ri a sc rid separate e s per iiien t.In paalel %kirthi the ltureV ita.1itLIg F\1-I4) medium

iii'cih.in 1 dth'(H ;hu' nuL'aan/ 'raarid 20"4'. fetal Calf serumTI. %%e set I l L culture cc th Rll\NIF-C )re %ceek atter Iocu.laling ttric ells, into culture11- flasks P,4(1 mIumtILI .uple -ild ccll itf w ii\tui of IT)",, horse

111 riti11 anl adheret-Cl Ccii Ia'. 111r. 0% marrircc C cutre %cre re- SerumIII aid (I 1)'o. fetal call' setiurn I he flask csa rcCarged

t a t e; !-cd %\ i l n I a l C (IL i l 1 n u m b er) C f tIn a i l W Fc~ Ic~ ii li imc dit i r n i % C eC k %\ c itt ' - 1 1 1 1 \ N I t ln i e s a il e nla r r o c sthe ani tilitIO%% alitfaiol. Flacli flask conitained a drfkererit aspir-atliri cOrtarriirig ;1I 1 l( it -( Ni1 D'111 )i irg Sr -sCek

icitlireC ilielhLIl. either RPAII- I104H. NIc( 'o%', i V\ Fiscliers. cuIIltre period, a total of ortlc lf'liii F I I-( Al \cere giroccn

,Ir alnI-aIR VIdun Mi or niectia .%crtc sUllpeiinted %\ ith iI Oi filte 1 oriadheretitl kcll fractiiri I l' result has been1, 1. hi)'W Se III i'fl l friri the n1 saute Ait \ ceklc rnterals. confirmed~ In a second separate kJCI 111CMrrln lased il theseir'ifacflCTi1ret-1 1 cells screciUnied aid plated Ili (l 1- -(IN1 as- etrilnt result. \Cc deckided to use R< F\Il- 6(.1 midiurii

F,~ f ck irtcrikcit csperitlietis%%cer pertornied. R P\1F- suLeil~)1ICtIed \% itfi 20",, prescrtie-Id tOIiits sli IrI Il the 1 il-1 -41 1 !-i \1,( 0%.- ~- niedlrti asC CkelUall\ goord resUlts. lOiic ri CsperileTlIts.

Nall ,i1imcdi In t\ci (if the 1ti'l crrnleilts thle highest suinI (c:c IF I -( N\1 citits1l lasks Conitaininig alpha niediunim o wrfisi id il/Iu'ilI '1t01'11011' /iMbii'', 'i

'CTci t11itter I N d ,- -M .. .. :- *ll. li k F I -C NI counts inorif itic 1 rthe Ikc ucsPciiilCtiS, MCs eras I isehier's riied1iui [tie etlect of tidocOirrIINOT o)il Fl -(l NI nmbe Inl long-sit i-cc the lck cst or second lossm es C -[-( IM couints in atl tfll rAIllarri*c cukil nr1C ss as, ',I urdd C)Iecekatretbihn

414 I speri nien al Ilenatologs sol . 17 II198~9)

I able 5;. I mipaltsori W ditcin Im tIIIIII (o1 ilIdIrros cell, usedL or rtch;irging canine tong-term fliarriss culture%

00 1 h I o 0 1 20 101 24!) 10,NI\( Nwst MMN hoost NIMN boost NIN( hoiist N1IN( boost

ad 1wc - ad hev - iher - ad her - adher-et ICIt ci e r. ! ell

clk 1 ( 1Ft - celk I [ cells (Ft- cells CHA cells Hfl

I1 , I,1( ;tlt 16.906 1 S-to 4V'4 NI 7) 10.Ax11 4 41 11 1 3;4 1 8.4t) S 0 24.30)1 146~ 68,1318

44is3 4'Ml 8 166 1o3.4-8 I8 V.(,444's I ~) S1821 26i t)"4 87 39,81 1

I''! 'S 1,5 19)' 1" -5) 3 4164' K ' IS 44LI 211S) - t2! 2" 14-4 17 Sit"

25 'i I '.3 Io 10 S .,()2 2 1" 3.74 1 8 1tr 57252

111110% 4t Is."1 4 6 34

Il ha ,jtLs 5R ihCkd I ssk hcl, 's,l.taIl IshtI I all ad lterelni cll o r~ I '.\ei [ 17. 30. W). I1210, or 240) 10 NI N( cc:, a in ing .,'9'7 .

Jn adhe(I nt ce ,Ll l er .I, ak,,% siro tchIa Irged \NI It h he sam nie adherentI lasecr or No, hether the\ canl bie allogeneic. DI -A-ilon-)um1)ber vok maIrrIsI cl 4I I N f4 VI I c ont ia i Ing 27.40 idetiical. ( u11llures %\ere starte[d b\c uilIIatl'g 6 0 I) N

-, t I( NI I ho tc satIitc ia r ro %\ asp1 iat ionT ( 'ulIt ur I - irom tile same mar'--o, aspiration in cultureC flasks. Flasksd R II-10 4tl ) pls 2) horse seru nil ssV aIssd either ss cre either not recharged or boosted after 1 . 2. or 3 weeks

55 Ibm)l ltd rocormtsI one orI suppl~clletntd ss thl 1)A5. (1.. 02. ss ith 2 -I10 MN'dens ed eithler fromn thle samtie donor usedov 11.4 ,is \ h~doots Ne." shoss n in Vable 4. the sum ofi or)I establishing the stromnal cell laser or from a [)LA-rtoni-ss cek s( F I -( NI counts in the noriadhereritLcell f raction ss as denltical un related donor. Results, are shosn t in Figure Iii~ letoir thle flask s it hoot :isdrocort isone. lThis has been W\hen Cult ures ss\ere recharged I ss eek after establishling the

c nhm c tid toI a sc~o tnil t ipedetil t \pei merict (data no~t St romal las er. the flask recharged s\kilth aUtologous tnarrossThos ti% 1). shlossed a h19iher Suml- of sseekls C Ft '-( il NCounts in the non-

ad herent eellI fraction compared to thle flask boosted wNith[ )LA-notiidentical marross (at sseek 2)). the ss ek het , re the

o'O O jfh' 1 'l! ,llttilt'' f lt~ll'''Ifirst flask became contaminated. 796.11- compared toI'S.))14). Esetal u inut thle first 5 s'%eeks. the seekl\

l ong-termn Mtt-arrLs cultures, \Nere started arid tteit rcharuted (1-1,'( coutnt T.~.e in thle flask recharged sN ith l)LA-aler I ss cek ss th iether I S II W, 1t). 6))- 10'. 12)) nonidentical man . pared to tile flask boosted w\ith

lo)'. or 241) ,It)' mnonuucar marrowo bufls coat cells aUtologoos, marros-0\ Os erall. thle suml of' the sseekl\tainedlC~ fromn thle same mnarross aspirat ion. The sum) of the ( J I '-(~N coo tIS in the flask boosted ss, itli DILA--nonitden tical

sseek Is ( f- IM counts in thle nonadheretit eellI fractton ss as ntarros 's \as as loss as, the control flask that s\as not recharged.highest i the lask recharged s\kith 24))- I))' marross cells II ricontras't. culture flasks recharged 2 or 3 ss eks alter es-I lable i. 11155 eser. s hen cottipared to (lie nlumber of'(Ft'- tablishing the sitromal la~ er shossed a similar sum of sseeklsIM inoculated into the Culture flasks at the time of' thle (T '-( i N I counts MOinpedent o1' ss hethrer boosted ssiih au-

niarr ss% boost, most ( Fl. t-( I s %k re obtai ned from the flask tologous or 1)1...-tion identical marross, eellIs. As shoss n inrcltartci %k i th IS ,' r marriiss cells. Af ter () \Aceks. 410~' Figure I. the sumn ofssNeekls (Ft '-( iN~at \Neck 21)was 4)113 140 th." -' ot hr iii(Hl-( NI contained in the miarross boost ss het; the flask ss os rechargedI 2 sseeks iiici c',ahlishittg thesseie ta))ined f'romi the ', tIa ik 1cted'st1 I 5 10)' niarro adherenit cell laser ssihlliHOIiOuW niarro s' cells, comparedclls Lonpared to13'b in tILe flasks boosted s ith .)-240 to 4X3 177 ( Ft'-C N colonies, ss\hen rcharged ss ith DL.A-

SItI' iarrmm clls. th is finrd inrg has been eonfimed in a noidenitical marross. \\ tuen boost ing IIe, . h..flask,; afterseCL~ntI ideperiden experimient. ss \ceks, ssithI autologous% tiarross eel Is. tile sum oif' ssek Is

I t -(Il at. sseek 20I s\'s _ 13.52') c~ompared ito I 7j1 39

ICO IW'Ial"o~t 1/. -1,111(011(d Mdrol ''MResltIsIpresenited in F~igure I also sliiiss that 'H-GicatlTI ~lt/5'i't 11. lirlit('liti~/ 11,/0 ri/lie liars ested front catnine long-terti rnarross cultures, b'r a

I he hlossmiiig esperinint ssere, done In order to fitid Out prolonged period of time. We kept scei encultulres until ton1-ss Ie11tlte ron sclls, uIsed to re liargC long-term titarross tamination occurred atnd sse Could obtaitn ('H '- oIiut of'cultu11res tneed toI( he aIllllgOiu% Lolnipared to tile cells 01' thle the itotadhecretit cell fraction foi' 2))t 131 ssks,

1 0 Schueriug ct a I ong-terin (antie NIarro r uliure 415

900000O

-,000000 I~ onadierent cell raction of canine long-terniiarro%% cultures recharged %%ith aUtiilogous Or a]-

600000 logeneic D)LA-nionrdentical marro" cells aftc; 1.12. or I "eecks ( ultures Acere started hx incubating

S50000V~6 -0' N from the samne marrok% aspiration

1 ii '5-cmn cantet -ncck Ilask at 2 - 10' NN nil4000001in RP%11-164() nidi uoi supplemeinted n nh 101,

piescreenecd heat-inacts ated horse serumn. Flasks300000~ %crc either not recharged (*) or hoosted aftecr I.

2,or I %%ecks %%ith 21Ii MN( dens cd eitherE 2000001 fri in the samnie donor as Used [o r estabhii shinrg thle

V) stromral celi lax er (C. I -s% eck hoost- ' 2-s\ eck00000~ boost. anrd I_ 3-week hoost I r fronm a t)LA -nnri-

identical unrelated donor (0. 1 - %cck hooist, A._________________________________________________________________ 2 seck boost: arid S. 3-sseck hoost). At seekls

0 2 4 6 8 3 2 14 16 18 20 22 24 26 28 30 32 itters als nonadherent cells kssc plated in tripli-cate in CT Ft-(GM assa% Each poii represent-, the

Weeks curiitit is ribier of keekls (Ft *-Ci NI

/Iraru,111r/ aalv,, o calow mi wrmand then to recharge the cultures with fresh marross Anotherlou ~ri~tt(pal~inltsiif i~it~ii t'actor crucial 1or murine as well as canine cultures is to ftnd

the right lot off horse serum that supports culture growth. Yet(antne long-term tiiarrtiss Cultures fortmlia dense adherent Culture 7-onditions for canine miarrow cells diffier in severalcell laxer relatixel kiJqL;l\. on]% I %scek atler inoculating aspects from the oinc f-olnd to give best results in murinemitmos cells ii-to the Culture flask. I. ItrastrUCtural analysis lutng-term Cuiltures. Canine cultures give best results. i.e..of the adherent cell laser 1-3 ssecks after starting the culture highs nubro l-IM whnicbtda 7Cad

sliossed that the diominatit cells wecre macrophages. The see- show no or ver% little (Ft-GMI production when kept atond most freqluent cell t\.pe seen vsas adipocxtes containing 330C. In contrast, cultures (if' mUirine niarro\% produce moreOuilenICRLis fait lropcts in the cxtoplasm. C lose to or ini direct (Ft -GMI as well as (Ft VS Ah len incubated att 330(' com-con .:Ict %k ith mnacrophagecs ssere hemnopoietic cells at different pared to 370C [1). In human long-terri cultures. an incuba-stages of mat oration. tioin temperature oif 33'C also seenis to be superioir to 37VC

in terms ' (Fti-GM1 production [5). Coulombcl et at. re-ported eqtiAally good results in -stablishing human long-term

Dic~~lCultures as judged by recoveries oif erN throid burst-f -ormingunits tl3Ft -F) and (TI-GM, "hen incubating marrow cells

In addition tor thetr use as a s, stemi in ss hich to stud,, the tor the first 3-4 days at 37C. and then at 33o( for the re-rCgUlatoiri ofhemopoiesis. long-term canine marrow, cultures maining culture period [14]. Long-term cultures of Sy .rianssere orf interest to us for tssu reasotns. First. this culture hamster [3] and Tupaja iAk [4] marrow cells grow. best atmethod has been suggested as a means, ofdepleting malignant 37C. as judged by progenitor recoivcries. similar to caninecells from tht, marrhis [6-S]. [)ogs svith spontaneous non- marrosv. It is unclear \It\ these species differenices; with re-fitodg-kins ix mnph~ii ep~resent apotentialx important model gard to oiptinmal culture temperature exist. Incubating mouses~steit in %% hih the clinlial utIlitx of this approrach Could be triarross cells. at 33'U improvecs the dle\ elopmencrt of the stro-tested (91, Second. keeping ma- ross Cells in long-term cultures mal adherent layer [Ill. Because the adherent layer is knowNnIcd icpeatedix ss ith \ irus-containing supkernatant could allow tot be important for suppoirting long-term hemopoiesis. thisai stficilent increase in the exposure of marross to retrov iral ma\ explain the better rvsuilts seen when incubating nturine

cti rs arid Ii uIs increase the etficienc% of retros iral gene Cultures at 33'C. In canine long-term cultures. grosvth ofIransler inti anine! marriis cells. Indeeud. in recent experi- adherent cells was better at 37C. which again ma\ explainmen11P %%s S esitss d thtat %%C eCould increase the efl'iciencN of' the superioritx (if the Cuilture results seen at 370. But ingenc transl er h% culturing marriiss c,,ls for 24 h suith virus- humian Culture. svhereas 37C( favors deveclopment of' thepd uciL ng pam kaging cells, and( then fuir ariother 6i das s in stroial adhecrent la~er [8]. 33'( seemis to be superioir in termsling-term L-.Urc tled ss ith s irusll supernatant) compared ton of'supporting long-term hemnopoiesis. Culture te mperature.res ults obtained \Aith the 24-h cocultivation olnly 1 fill'r ho~r '~b .Th'- addit, ,, l !-,tors Iivi.-

I n 'I LiI.F" 1-1 Uw'r; h ii tltis siud., alloAs canine % ant fir the qualitx (if long-term miarrow :-ultures.htco P nen.prcirsiir cells to he gross n tn long-term marrovs The groxsth medium cons entiiinallv used in murine long-

tultture rcgularls for 20tir 31 wxeeks. These Culture conditions terni niarrrss Cultures, is Fischer's nmedium [ I] but Mc( oNsarc bised 'iii findings first niade in niurine long-term cultures 5A- or alpha medium can be substituted it 5. Syrian hamster

I I iilar I,) thi- Lross h reqlutremnts origintullx. descri beud mirrois grrrss s best in RPM 1-1641 medi um [31 and iuprainIti riM, il IM'ic- term1 stiltlu res. As ilals() fond it ton be i m- miarross has been cultured in Fischer\ iiecdiuni [41. The growkth

pIi itlt I'l rr ;ti c ull ores to !irst establis tin adherent laser mcdi Uti for hiumnit long-ternm Culturcs can be alpha medium

.4I 1 , xperimiental tli'flattologs %oii. 17 II19h9)

(141. but Fischer's [51 and %le('o, *s 5A [1 h1 medium has e One of the problems faced %% hen setting up %everal long-also beenI Luset. InI Contrast to 1t1o,111e and I opaia mlarrok, terml Cultures f'roml the samne dog is to obtain enough marrow%can ine niarruss cells did not gr-os ssell in Fischer's mredium,11 cells I .%cek later for recharging the cultures with autologousBest result', s crc obtained in cit ficr RPM I - 1 h40 or \lc('(ix's mal ross cells. We thereflore comnpared long-term cultures) \ tdIICILimI boosted \%ith miarrow cells that vWere ciih.-r autologous or

As mnltjund abox C. thle SLIcceSS 01 rineFF aS ssell as allogeneic t)LA-nionidentical compared to thle cells of thecanI~l inc ln-term1 cUltureS is dependent onl thle gross t-sup- strmal la\ er. Cultures boosted after I \%seek w. ih DLA-non-lli'rtiniz_ quLalities Of selected lots of horse serutti. (ienegr ideroical mnarrow cells contained less ( F['-( i than those

['Ireported that in murine cultures the qlualits of,~ei chtarged w\ith aultologous marro%\. but the differenlce betweencicnit' lots of1 horse serr m 'ld be imipro cr1 h\ supple- autologoIrs and allogeneic culltures disappear -d when re-ItteittinIL! theml \\Titl gluococorticord. \ddition of' 0 " MI h\ - charged 2 iir 3 sseeks after establishing thle s, omnal laser.drocortisone Increased the ntiimber of adipiicx tes as ssell as This miax be due to thle inabilit iif" the culture sx stem tothe- rotal adherient and nlonadhCerent populations. lDester et Support SLTr\ ixal and prolilcration of'T-Is mphocx tes or otherALlI 'and that horse serum.11 batches di 11cr ss dlcx\ it their level cell tx pes that canl esert anl i nh ibitiirs efliect oil tile DLtA-(if free corticosteroids [ 18]. Serum ss\ith a ;clatis. cl\ loss cur- nonlidentical cells of' the miarross boost. Thie results of' allo-

costetroid les el s' as insariabl\ poor in maintaining hemno- geIleiC Cultures, recharged after 2 or 3 sseeks are similar topoiesis. ss hereas sertim %\ Itil a relatiscl\ high level showedc observationsnmade bx Iexter et al. [21] and Mloore and [)ex-

crOosst-uprigqaiis \\hecn -'piol- batches %were ter [22] in murine long-term cultures demonstrating that ma-stippjleIInttd \\titlt hdrocortisone., their lUialit\ imnprov.ed lor . ii-2 dIiiirences do ttoltimpaiir the caipacit of the adherentcoiisiderablx in !erms'of'long-termn clturIe hem~lopoiesis. Thte mIicr-oens ironmenC~t to stanallogeneic Stemn Cell prOlifera-gross, 0it oltranuitI-termTl niarros Cultures is also i11- tiol_

pr' i, 5 %hen I MI Itsdrucortisrie is addedL miaml\ be- In sumnmars . ss e ha se described Cultutre ciind itions !fatCa' ,C of imipros ed rfeselopment iifthe adherent lax er [5]. In) alloss fotr the growth iif catnine ntarross cells in long-itrmc it trast to Itlo te I1% it 111LCItCC iof*t hlriicirti sone on mlu- Ctiltutre. (F1G -CiIcolon1ies Coiuld be r-CgtrlarI\ grossi (iut (if'11i1 itea d hiL1manl l~ ng-imltltrs \Xc I'utittd best restilts in the nonadherent cell fraction f'or 201-31 \xeeks. Wec are cur-hose cani teC long-term CLIII ore-s tltat \\ere nut1 supplemented rent\ Stodx inig boss long P1 tipotettt sten cell', stirs i x in

xx i Ith x rhO 'coirti site. \\ i tt tlte add itioun of crirticosteroid to can inte long-term Ctiltuires h\ transplanttintg a tt~lOgOus mtar-Gilt11h o eCi uhres, a duse-ufpendent decrease in ssekl' (IFt- rosy cells kept in IOttg-terrtt Cultures, for diffierent periods (of'

IM it .1L t hr %\ as obsers ed. I OngL-tertit Cutilures of Ss riart i rite intto letliallx irradiated mnarross donors. AlIso the efli'tctsbanster [3. 1'1)) or Jiupuil 1u 4. 21 ntarross as skcll do not of added hemnopoictic grosst I actors oil Ithe lunges it lxOfreqtLIIC eisppitticnitatitn ssith It idrocorrtisone. p1 tipotent stert cclkI ini cartinc long-terni Cultures are being

TheC reasons fur tisl' species ufIlireitee tire tinkrItos i. In studied. The results of' these esperimerits ntas be reles anttItiin alHInd huimait long-t erm CLti[rces. tsdriicortisone stint- Iirr a nurnber if'sttidies. inclurding purging attilogitti niar-

LilatC 1Cs t\ ilex el'pttt Of Strorital eellIs artd adi poex tes. Botht ross grafts if dogs, sith spontaneous Ix mphomat and fIrm In-ocll ix)C pes ar Ih Itotigt to be iiIt'antfur eStabl ishirtg and creasinrg the eficienc (If' relrox iral-niediated gene transfer1otainlairting11 teiCiPriet1Call% acts eittrine antI hiurnta long- itto canirtr Iteriopoietic Steitt cells.IeiiI cdltires. Bone niarrius tlttire,, front te Ss riat ltarttsterprl 'diic sterit cells tiat priili ferate and difthreittiate for - 121 Acknow ledgmentskosecks In the absence II an adherent layer 13]. The fact that \,\ thaitk D~r Paul Sintoits for helpful ads iec artd distixsmli. aridIitstCr lOiig_-tIr CLItirCS can gross ss ithiiut a stroiral laser fi(rbn.Rbr al ,Cadsl .uxCiuitrrg.Iirnxcsiihii %s Itx ithe\ also) 'i ritot retilire Stipplementaition \iishurit. Wall\ Nlexr. antd the Slieu Wkord PrOccssirtg D~epart-

,k ith Its rrllcrtiSllnc. ssltrclt stliittlates tlte tles elopment of ieni foir expert teehnical ;issisiallee. Tit ssork xskis supported inart aitrctil cell laser. In contrast. bo~th Iitpai and canine ixirt by (;raitts ( - -'i4. (A I\ 1,5.s (A \ is221 anid ( A 3178

IO114-tl~l nW~rO\ IUICSCIC)C~d Ol tle resnceof'ail asstirdic h\ rthe Natlional Caitcer Instittel. I[[ ll.34 alarded h\a l r r i g -i c r i i n ia ,,r i s c i t r e t o p it o % O t l p r e s e n c e ii a rt l tile N a ti o ita l f i ea l h . t ol n g ti t d l31 0 ( 0~t l iI ~ ti ii . N i itn a l I n s tIt o re s

atficirttLellaxer ut eeml~rgrss s ilirit tipileeiiatiiit Il Health. D~eparitment of' icalth aitd Humiiait sersices, and b\ theIi di r Lrts~ nc. The adlitren t litntoplocien Ilt icrten\ si ron- \rnted Forces Ratirobilllllg Research Ins'tiluret. l)eficns Nu ucleair

ielt (,I lIuj'oie long-termt Cuiltuire% sltiSsasat specific char- \geitux. under contract l)N \-i(i I -x( -M,)"12

acitcrixtic ai perilrio. fciachmitit of adhecrent cells at inters alx()f I -_ 2 il tths hI I lus d bs xubsccu tie rcgeneratritn of' the ReferencesadlrcII iti el laxer [4]1 Itis iricr'ttrs e ctpacitx of-the adl- I.tciiI\. ient)tataI I'ihClldiitrttrll

icreril nin. rC1rts irliitnCIII! SeCiti111 h e Stffiient for main- ling ilil prlifertion (it- Il liNlle icslit cells, in \ itrl. .1 t elIau1i1111 he11 I011' IMLi-rr Ct~ltore's. tlCtch% reitreriig ad- I'lill 911)Is

ritIll1 )'I hdi i c I iteT1 tinnecessarx . lTh gloss h of cartine 2.Sakakeen\ MA. Cireenbenger IS I 9N2 I ( raniililpoiesx Ion-has ber) IT-Olf~k(fCPIIL]tlt il crtai its1 M r lon tr1tilLIs hb:Ine ttarruss cu.1ltes arnd fartir-rfepeit-

ne~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~kci tcntc('i' tsbctsrllgxreetfrt(i eti ell lite gentertionit sigitilicait sarlatiln amonllrg 'S InbrerdIts,( hIr scruir.1 It cLi~iA thllrlc~~c be that xs hen srreeinrg rilolls ,trainit, antt IlilihIed i S~lckS I \all ( ancer Inst 68:3fi5diliferi llsw,f )thorse scrumn. s\c %sere selecting fbr ttose "tsl I tsint F.. lDnh'iln I . Katsnclsonl 1. -\ritlr E. 1511 PoPla relatiscl% high I.IrrlStcroil c l. \dding hx rrocortisone Il HSX2i i I ItI- PrlililrralI rl ifleltilllliei ltii cells in the

ill t1e iidlir'Cs LIld t1t1s it\(' eInIc(reaSCr thle Cirticlusterrid abserLe I art adherent Iltonola\ er Hiti~d (,Ii: 1 301-4 Mbirc %t VNs. Sherrdarn NP P. \len 11). [)(,xter FV (I1i97'))

it\Cs 1) i he tIr tiltorCs litt' a tu\ic rartg-C cas"itig rnthibitiin if' Piolllnid IierittupuICs1 it a lprirlate hlle nittrr uilture %ss-,uIlturC gruss lb. %\Ne riot111 test the titlit\ ofiht drocortrsone tL11, . IdtaS ti(S ()I sleitI tell produiiionr antd the hemato-

addiltins ill stibuptintal lots (if blrse SCRuIMi pwllrillcrillI IrnttnItIl ld 5

S,- 11 ChL~fIflk n ' I gct I o~ng-w~im ( ann Nlarro% tltutQ 417

k .1011, 1 " KJ11~1A I l ' " I) I L ! -teI CIII Lilt II C It Umni I , A -hiahanil III M . I C% Ile \S (L (k) \1io1LeUenlr Noill hc-Ne nixl!m L-11 PwII \,i I 1ai \,ad ii S \ - -;t mopiiis. Ne" Ywork- P'lenum. p 9

f~d I I [[Le11 I I (00\01 01 l 111t 11 ' i ICIalICI :tiipit c lk 14. ( M110111110 I , a V.\ Fa'.cs ( J ( 98~3) hwIn ,ttic treat-III HjlICN Of hOflI I1.1 'I% 110111 Ieikclmii like. I Uit .1 ( JHiier 111nttIll ~ig-telIItI hLItimal IiilaffOiL 1.1ltures1 ( a ls i prefer-

11)1 IIcol 11 41 1 enia!. lcation ofI priml:%cs heinpojetiC prollntul Inl the' ad-him- iJo I.\lrciI',tern (1, lieAko 1), 1e~ta \Mi. ( [Itlilith I [Iherent laser. Blood 0 9:')

'i J 1e I x II ll!im~in I )xiei [lI \1 10oso Reei'nsiituni o I *;. Spooneer F. [)ester IA!I ( 984) Long-term hone marioA cul-ii J ei C, ?II 1) ,1C[I Ie' 01t1 Ji aini uo tma1n;nl lJ ta kenI IT'Inglt Fe- ILure.. 13IiIC [[aeIMato 48 366

ii ol m\el ']a 1CLeikwiem Ind III nin Lon-lerni 1 i. Gireenberg HM. Nesshuiger P1K. Parker I %I Nosak [. C ireen-u liuic I n.t Ci '-4 hererC 'IS ( I 'IS I ) I 9Min rnUloe\ les gericrated Ini cimnUOUS

N .Ll"Iilhel iKaIlois, K I )Kia, 's. ( opt. ( M. I I\oVI)IC ii~Ironu( c'UlTre X, jplI\ sologreall\ nolrmial. Ili i:7141',1 1 oiil-tell 0111111)%k Lliwic res .. ilk V.l iosnal n r (ireenherger IS ( 19 6 1 Senti 11\ )s*I'orti(,otcrii-dependlent

13,11 linillp IC!II. poCi!III1 cll e I II pat ii111 %i Ph lkdcl ph Ia t ii-l isa tIp~eei Ililarrol preadipfllli \ leN oi(hestItO ~ ~ ~ ng I[eiCiiiEkna.NWI dKII1ak'u (it) Lit)) ric. Natre , ' '

\L~~I ''' 'IS. D)e\ter IN! SpoOTneer F. Simnm ons P1. A\lcen TD ) I-S-1) Ilong-\ppl' iam I R I~~ II1 Slotrh Rl Secll t tharn I ( s a.lc ltr Il IXInarok cluture: ani o\ c' \ Ci\ 01 ICe 1111ILIL'NXi .ii "I~pcO l.

1~~~~~~~~~~~~~~ ijer k\ I Whl I~ XT' 1- cClillIt IFdiS[idg i [i\riltD Ij1nbI-eI IS lid%) [In(g-1lr Ilite marro\&iiiI'.lihim I~flpI.fl~liiil 300c1.lture1. Ne\% YOrk: -\1a~i R 1 1)5 P -

kook L\\ .hrICIneI I . Stead RI!. \lr \I) I I SO) Rtiro- 1 1). Fasirnent (T. RuISeetti F-V. Denholm F_ Kat/ncklsn 1, ArnoldiIIlalll t cnic, iii1 ,Inlnc 1teinI'pnieicl IrI-ogelitiil cells I 5 P( 1 "' P I 9,S ) ei tue1IIC % [ I Ili\d L01011\-11o tiling cells

I \ 4, - In longi-terim haster I't MClMatto%% suspen-ton cultures: re-

I~ h.I C I!ii It 1! 1 .I IIIC I N I \\ I 153 ) 1npo k d CuL lure spmlse to po0kessed plee itditiincd ItiediIti.I Blood 601:40'o Lartt,,tcmaiph p,,cnto Ms[ahstri. Mo Ntore NIAS. Sheridan \P( (I1 1)"9) PlUriplenni;. ,tL ni cell

h !iI'C .1 I 'Nih1 R C 'u-t C S.\ICCIA IIII It I . l)e I I 1luIM 1\I 1 u LtiteC. 11100id ( eIN i'29V

'Co1l I IN t ( ii 1111 ILiI~Ie1II1 1.1LIM~t Iiiiiiiiic~l teillsdI 21. Desier TN!. Mioore \1IAS. Sheridan AN ( 11I 'f7) Mlain tenance,ind ( II I -(\I s 1 I-like antigen' aN iciogntid h\ isko ofi lieiiopoici Niefl els , nI)!ii KI eion illd ftliteeiii.atid prog-

!! I t,'" I I -nI 11 t moll " 1, 'il i lt Itt! le. Bllooid Owi 10 ps en% Ii allogeneit and '. to allogetic i ne nlaiross cl. mecras ti

m Seitni , Stwob R. Nash R. Stecad kii. Kilik \\.\A . %fiIler s ro. .1 Fsp MceuI 145;: I 1\I) I 'I Ri ii i ti;tnsli ml gci~nc ilt' Canine lic-main- 2 2. Moore MIAS. [)esier I \1 I 97M.8 Stem cell recgulation in con-

p-i.-!. ~ ~ ~ ~ ~ ~ V 11i'- l.I'.Ii i~'i I/mi F). \scvi.'ao iIIIN' lIIieflltlIilIi -CII c1lItur. - Ii~il~lIli ['roe M (83

J. R D1I . R1 ',., 2). 255- RA4I 1119881 ARMEO FORCES RAOIO@IOL.OGYRESEARCH INSTITUTE

SCIENTIFIC REPORT

SR89-24

Radioprotedion of MIouse I-emalopoielic Stem Cells hN

Leukotriene A 4 and ILipoxin B.

I I 10\I*\S I . \\ \1I N, I. k.

tB LI . \tITIr ~ -[,1, 2)1 4 I 14

IRu(iIoprolectiaI) I .tikolricne 'k, I ipom~n 11, 1 r1dI),Lnoi, pluen cuiio formiaioun Mi,c hemaiopmiuki

'Ivei cull

k -1..ClIE I T~T!II~I lC ' ' II Cl 1\C 'I I 1i l %\1 1 2 i ,I Li. I K

T i l. 'v i !I, 10t ll C l' III 111\ 'l' N~itI 'Il CIICC'' 'U111 A' M!iX IT ,t I CA\1

I VIROI1UCI I0ON

[tiec\t 1cIc propcrtics 01 cicoaniid, \\ere ticd tor both c\cfo 0\\Lnc piixe Ph fact

dild. 1110!C -ccttj irlpO\\r~, 1 na 11podUCt' to inldUce aipocio 1ti "1' jlcd~~ ion

,ho~ n \jltl ccflk inl cuhture- 1111.'C heritatopoictic ,tell) cclkil Ill-() I) and \\ itf Cx hoic anlill

Llr~ i~all K [RFI of 1' oi arcatr call be obtained \6th01 l~il- a "\1niheac cI i ati\e of (ticniatnraIl\ okcc nringi prostaglandin F.,, anid v kih I.F .4 1liesc cr toptotectt\ r

propcrties ha\e Nignificaitce ill normal phvsiolouicaI pioccNc' and also ill caltic Hioloe %\here

oltte tnlmor,, produce ele\ ated fec el of cicoquioids" an'd 11W\ inluenLcefCra peC )tit iC efcc

Pretrecatment %iil 1.1(4 pro% ides a DRI: of 2.01 for protectiont of MOUN~C LIauIuufiocr t-

m lacrophage progenitor ,teril celk. Because of tfw ,ignificanit DRI hr. this otie Iipoxrgena',c

product, hei potetial for other protect ie lipo\\Lenase productN tkILnIngI the llrdro\\LI fatiacidc

and fipoxins. ss a, examined. ILipoxin, are dlerked thr-ouLh the interciitionl 01' 11ore- tftdtl one tr peof* lpoxr ueiase Aft houih the\ m~ar be formed hr ,cs era I hiosut het ic ron tes. tire best

1hna~i/"il pallmx\,i\ eI AC li Th eu eol M'a '-lie\%cCfiaNC err Ia I )0\gLC IitleI" O~idiiI . clI 1hci

1-iI111 II'o er , III IF . I hic 1%%o Ilrifiia\ Iiaill x OrCcii HIc 11110\11, Irc I \4al I \ 134.

I 1110\111 nrs'dnc,:renI 11,1' 1,ccir dcliwri~Ille TIT Irnnanl nlirefti il\'', ral lxraenhilic Icnkciliih

.1 11Illreicr k ac, 1 1:10110 'l 1 rit 10C\ I C' . I lic o n~cnIIiaI tild t It ofrici l\ C Illi tICi ,c ot I I,

\cll 't,~ Tha i I I a. prlen:r~ol o 1 01c Iinemiii' uld tlirkei icc' 0i) hliihuiIicih. 'icnII :cIt

1t (I I I nc. prc'cictd illit ln ,Ipci.

Mt kI VAINXI "s , ) Mt1. I I IM)s

!oi Rwi\Ie! Kcsceir,,h I aheir itoi\ Wh'IiladLclia. P' *\I. \taciidoiic acidl %% as ehiruird frm \1i

11A I'rcn. III,. H k\ 'rai, M1\ I. I I \ . icih\ I c\ic\a, ihc c mcreim i o'V [I , .Ie'.li kekacli

\Icr[ ,k I I o-t thol 'Ito it." P !,I Ic I Irc- I )o \ at ( I Iud I hI itIIcc aill ei I I \ . \\,I i cilau cLI

I! Il !lc. iIrli\% I Cltci 1'\~ ,iui tiat e io iii i ha 'o oedilili 1di eoidc 0 ) I. ~ \ err 'c

1"! -4 hnn. I he [11Ill ~Ia hci IdLIr.ICl 10 -. 0 \%Tii iln111C I l( 1. 1 acli criimiold \i\ a' c~ i~ioicdi

!,r 1\11C-' iLIJLidi ilIIIrecCII 11d CiiCdrcnc cIl iiCiharie. I-iarl (1e1liiei' theren aiplpi c

ollillfillanl \\L'Ic aIdll'c ti t) 0 .41" Clihiiod ill ilnks' hillicicl 'all oltiieiiI'.l

eliclI ed'raiCi'. ( filand WIld. \ M otiillpoiid' \Ccc dririndh hn~x~

inIL Ict.110ii IlL h rlilpt: o Th leiick ill a ieoliic of IT 11 15 IV iln hC10IL iirliriiieii. PICk eui,

lidL'Ilt:s Ilcikli it L' h optuimial Adiiii1i11i11,010ui litle loi OIlICI dCtCsaI0Ikts iiIg' ilic I ( I L

lc"",i LI''ichL'Ll lL: I' 5 11111 i !of T if iradiatlieti;

iau Mlle ( )21 mial- 1i1icx. P)i) 12 \%ccks Ald. %kcrc ehrZriIrCd reom (Irarlc' Ri\ci

(kiic'ijoti. M' ). *\iiVifi'lkl \\iLIliiaiall!iicd ii I p"cried of 2 %kcc~k'iwd cvaniiicd lot paihol ieial

\\L'iL i'JiXL' W Ii) J i~~ ii I\1,0,001 rcrci'Llaic Orc'cili' e-cipcilir L'Ilin!iiiii~iilcd

fict flowi. \lj~c \tCIC IIIiiiticdl tit a 12-l li lni-diriik -%Je 111d a\imidcLI \%fill \\t\ ilL' Reoicl Iloe\

dieti (( oii iiclital (ai ( o., Ince.. ( liicac, 11 ) arid acr:d licdl \\,aic[ (01., aibl/ine;. V\ [ lic

lrrIdIUiiti \111c \%crc ii raitwd ill ia hilicral cohali -6(0 c~aminui ra~dliai~ eld al de'10C

tilel i (1 ill[ intno a ielia ll'' 2 dol (1 as, prc'iensI~ dc"cljhcd.

I1cl'el/Iit'l{ 'iec ('1/ kiatc. I- -( I I. forniationi ", ts,,iicd t% (lic mcitlod eif Jill midl

NIL" tilleuicL '. as rN ieil dcscrhcd' 13ricl].% niicc rccied '.5(1% ef irrdianen . Ilife\ \%crc

kilild i 101 di\ posuirraldlilaiei I \ auspli\ \iaieuit in chirged~ ( dcianihcri aind hiii snlccius

\ crcICi telIcd MidL 1;\CLI i Ilen1iri selitieril. fice liilhcr of celenlics pcr sNl)ccil r CICi & i.iiiiLLI.

aii( lic iieicaii and~ ianidard crie %~cic reporited. Sraili cail siL'uliaiulcc \\t', sLliriuicd hi, anialx is

eit x irliatiis in1 SinLCli i -ic'i I lic icneritcd iiiccsc iii I[-(I I iuiuci, arc r-cnrediuchlc.

RIF.St I PS

Effecti of dLA4.d LA , I cCI1 5-PTE.I)jII0 and AA on ICFU

)1 6 iLLI I \I: . )11 1k II , I t I(I ,,- .i ,Lto 4 It d 'c'2o

ul' l~c o~ I!!h I:L 1 0 IIL'111t, .\ LlIC ,11111 %~i 1)0111 d '\ 1. I! L

iT!,I I10I 111 ,c IIIwpI)l da I I tI ,I

C C Cd! 11 1 It11 13 ) III-111C A lllill 'M t1111 il\ 1 L II 1 11 W 11, C21101 6 -C)I N IL~ T[,

16 6

14

: 12

10 15 6 7 8 9 1

1u losni:D 8tt tciij~tjci~itjiuiu iiliii ~mi cii ci iu 2 i t'

itiiucrt i i li i tiihru -IIiccuccijic iidi Iipiti- llur

I 6 -I' TI0 ritlluid~ t td TA 0L[ciJ~J'iti~i~ii i lcitiILLi~v ri i h ia.(ii~ 1 A at li Iii~:ii ~n itiii tiiIi~

* *:~~~~' I I( ,rrrJ I 1 B c .J1M! T '

I' I II I . \ how- itr~ Inh~ ~Lr~r*t I *( 1 1: 'M 1 d , om pil I lp l l

I I ~~ I I III I I I I I[ 1 M ii Ir IIi I. i 'I 'n I n~~c

I , v- III 1 t I IjI ' II I c 111 L * It c j III I I 1 Ic I dI I t.1l l I I II I

1 1!1-1, 11 11iIi I\ I\ rj , ! nli L' I It\" I IlL11 I I tlc I i I n\\Tc I d'c jI I 1 cI d C\ I .I clL

2 ,I Ckl I X I lici. in tkIIlI loll. HOi J,I'nIIIll r C'ICLI I Il 0 1 0in\!k:11 Hdu llo \ 'ilhic LILLIlILl IllC11, 1

1H 1! \I n ' Itli i mc 11lL 1 itn 1 ( 10 111 po~i Ii tLaI lilit \ L C aIfIMpL iII

I Ilc mic . hll l ot ii ccnIl I zid&hontci loll Iiill tI icmcr i l i i'tll kiiX 11 h T ihc:

~ \ .1111 I~iLIIILLLI LhCiL'iI1111L,11i11C iit Ill ciiiII iciI1ci0 i \ IC iXhci ItIhIlILLI( Iii ~l C'LLhiod io% 1iin dilj~iLI I'\ L \I.I Ifol)1. Iikc tiU'i I~ c ti~alomI. ale \anlNk.dcii\ I Ilei\ din)

11if 11ill~IiLi c \VPI Ilunn l olnni hu i ii pliti anI l i, (1 priItell kuuiic ( I I \,- midl

I \jB -filLI .c wdlokpiuci:1 mil l \ li likirlnlil rcph)Ic throug'h dhickI I illIcilcc oil

lll\Cli let Icn kc I ic~pmnui~c to iiii CO~oll% uilnuunin2 Innchnv . Similar uICNc lor I V\ uili d maii

I urtficr rc~car-ctn. ')oiuic tumoikr :clk' pliodIucC I V.'\1 dull therefore, I .- lnLIHlkCC protccluoll Lkiilk

\[y~.:I: It WNrk I 1(1II\ T I ) I It VT\~ I T'LtiL Ii \k. ~i~T

J! I N, Ii:22W>''

itL, Nk. N:- 22: <

'I'WO- Il a

4 I.,:t~v,,t B K i IL i I 1itl 'I d K tL '; [ I i.~ ~LI i d itN .>I Kl~vii 1 1,ii Na, i I IIt TTt

2:! \ 4510 N

I\ \I 1) \

V. RX" I '2.I n

*: ItI\.s RNd !.I :1., -

k- 33: 4:'1 4'1

H 1i NR- w 134: 1flR 12-2

4,. M' i \ 2: -Ii

~It~rt'''jiieii NI * 4 I 4 '::~Ie~i~M: 'ui'J' "I 'Ii'Il\ :1'iui.2 :I2 ! fj '' 'i

'is: 'Ni <~'~ :''V'.x j,'' e :!L'~eU~ieI,. 2'.I. "440 I)4I{I'

h r iIio , IkI ,, I 1 )(1, . Iid e.Itou 1iin :]:~ '! IiUo~ tll I 4 ' tim 444OI ' 441

ARMED FORCES MAOOSI11OLOGY

RESEA ACH ITITUTt

SCIEN1IFIC PIEPOR1t

1142 SR89-25Selective association and transport of Carnpylobacu,rjejuni through N1 cells of' rabbhit

Peyer's patches

Rio tis\RI I \Is R.' FIN LisV So 1tt1\1 DI f01o( K. \ND) Jet 1V P\sii R

Ir Po, 1i , it Midi,' b i,i R o \t al' mu/)1111, lit the %Ia. AMl 'ON5/ 4 i/4S. I .\s I

\N I )

t ij/ %feli, al Re wptr In lsimric. li';jid.if/ l) .20N/4 5055. 1S .A

Recised Nlaih !1. fNOAccepted JuiiIe 7. I 058

\Si ki . R I .Si M Dtt~I R R(K Is . 1. A .2 \HR. R J .I. and lit R. 1) l9S8 SefiCLICde 0,11 ,isciiil iu tiipOf II n

hli ptlI/ion thromih %I cell, of rabbhit Pc,,cr\ pa~ifc (',tin J NI crohiol 34 1 142 1 147

MI cells inI the Pe\ er' patchexs t aci if ate I ranport of pathoeen'. ,uich ' I, Giipbocp ' t pjtJu t 01 the e'.u inIC 5 ces aluated th is h% pothe'.i' h\ uxLINI elcOton nincro'.Cop\ Oeain eesace.i iae dlt rabbit leal hiops. rocILAtedy. ith S 1nil - I ,'peIIsI0n'. of Il0' 011ni 111 (it /i C 1,o tr ptlunm. PC\er's patches tiken at inter\.i low IS m in it, ' Ii afterinoculation of loops. InI inae'.thetiued rabbits. pros idcd c\ ideike that (anob, ihour leUlii '.eleitit c allicred ti MI cell'. I,

uipl-t'.ed tii ab'.irptis' epithelial cell'. and "sas transpitd. apparettift Intact. Into the Mi Lcll follicle Althiuli intercflutlatoregini~ixi %%ere seen %k thin the follicle. ntanN others ,,kcrc phageoC\tosed h% l\ inphoid 1 11' the roinnt\111 of the Ikiiiphiticanld hiiodi circulators, is tenis. to the Mi cell follicle makes til, a probible rote for ,.s 'teimii spredd of ( Ioi)11o// Io ( /itlei

\A.I sl R-. R I., Si iiM -si ot H-(Hio K. F. A. P-RKI1 R. JI . , t 1it RH. 1) 19885 Selcis C i~ssOi,1ti011 1n1d trans.port of C( ooqs)Iohai-tur ieuwi throughl .NI cclls of rabbit Pcrspatches.. Can. J Nlterobiol 34 114' 1 147

)n croit queC iCs CeiILIs NI rresentes fan'. fes plaques. dIC Pet er p)ourriient lacil iter lit ilirat ion de path iuiticnc Linie(dp1oa cilfteii~r ljumn a partir de l'intestin Nouts asons i'salu& cette h~ pothese eii e\aiinait pai nicroscopie electroitqizife'. plaqlue, de' Peter Jce le lapin idulie par Lt ntctb'e dcs atisex ilZale' hLaturees1 ntocufeeC s e CC S nil1 -I111 due .u.pit'.IImnICOntenantf IW) cfli nilI.. L.es plaque'. dc Pe,,er prefesices enitre IS inun et 2 11 suiI\t ntinmoculatio11 des arise' Lte, IL' laIpinatlestfic sI' 11itt ilii e t as ec C% I dI' V ri Uc i ( rep / ta fr 1cliIIn ad he rc e ti eiIC i% IItt tn Au cell ilesIC \ 1 tit Ct quiiaui cc Itile'.epitheliales ab'.orbante'. 0i C(l .if itrainsporte appareiiitit Intact a i'intcrieur di licul deII Li elu NIU N1.tMnie I ides bac-teries se: retrilus in dan'. le' folli1cl plii'itICrs aUtre'. sont phaipii\ tci'' par les cellule'. lf tiphoidi'v Le follcule NI clittaproititite ifes x\ stinicx circnfatloite satteiii et Is niphatiquec. if deitit li s ii probable' de disseiinatinn s\ stetitque de'

amrp/I ,d)aj ter p-pati

I tradil par Ia ret, tie]I

Introduction 19~77. YoUssel et al. I19X5). It is possible that N1 cells could he\I cell,, are important ant gen-satupl ttg strtucture,, fotnin Itmportant in this process associated with pathogenesis as well

the epithel nal laser ovecr \Muphoid fo~llicles of the gastrointes- ats being keNcell eit r presentation of' foreign antigen to thefinal niucinsa t Sneller andi Stroher 1 986) . Thex, were ortoinali muco1,2sal imlmune s~stetll.

termd NI Is o NI ellsdue ~ ' We exatmined Peyer's patches (PP) irt ligated rabbit ilealtci Icells o icrot'OdCli (Ct thetr surface struc-lure Since the surfaces of* these cells have subsequently been loops because ktots ledge of- N1 cell - (antpvlobacter intrac-found to) he t ariable. the terni Micrololil has been tiropped. tions could contribute to understanding the pathogenesis ol' thebut the desiation NI iretained. Since their initial decscription disease as well as to the development of' oral vaccines to thistO\s-en and Jones 1974). these cells hateo been shown to inter- organism. Moreover. if' Cap slobacri does attach to and is

ac wtha umerof plhgcs.[-rexample. irocoea translocated by M cells. its unique spiral morphology could

attaches, to and is taken tip h\ M cells l(),.en et al. 1986). Ill provide an excellent model for the study of' M cell functioncontrast, strain RI)EC- I Ii'a/cricltia co/t, w hich causes diar- under a variety of' experimental conditions.rhea in rabbits, attaches to but is not transported through theM cell to the subepithelial Iii mphoid tissace rinman and Cantey Materials and methods1 983. Salmonella tvplti GIFU V)(100)7 adheres to M cell sur- C'ultivauion o/ ('ampvlobaeter

face ani dstrnys hem threb permitting entryv to deeper ('ampvlobaclerjunt strain H('. Penner seroty pe 27 I Walker et al.tissues antd to the general circulation I Kolhata ef al . 1986). 1996). was isolated fromt the blood of' an enteritis patient frort Beth-

('amnpvlohaururj juni is noiw recogniized as a major enteric esda, Mar-yland. Frozen stocks of the organism \xere thassei, inocu-pathogen that, among other virulence tcharaceteristics. can be lated in trvpicase soy blood agar plates. and incubated at 42"C ininvasive and has been found in blood cultures, from inf'ected plastic bags with an atnmosphere of 85% N,. 10'/ C0 2., and 57 0,.humans and animials (Blaser et al. 1984: D~rake et al. 1981: After 18 h the cells were suspended in bnicella broth supplementedLonghield et al. 1979: Spielman ef al. 1986). This may be a with 0.041/( eysteine and 0.25% serine to a concentration having anphenomenon associated with movement of- the organismus 01) at 625 nm 4f0.05. Six millilitres of the resulting suspension was

overlaid in 25 cm2' T flasks containing 4 miL of brucella blood agar.through the mucosa and prolif'eration in the lamina propria and After 18 h incubation at 37'C, the fluid portion of the biphasic cul-meseniteric lymph nodes (Butzler and Skirrow 1979: Skirrow ture system was collected and screened for contamination by examin-

ing the culture uinder dark- field microscopy. It was then pooled andAuthor to %~hon alt correspondence shouldl he addlressed. used directly for rabbit challenges.

Printed in ( ,,n.ij tp ti t (i

I [IIi. Icd ne aILl..)\ etl~liam'Tf'l~d~Ctt\11 .itlC 15 min incthl \iI1ti 01 llpp l clii mIAtu IA I k 111181

41

[-1( 2 Scanning clectron micrograph of sUrfacc ot- Pe'.er patch follicle after 15 min incubation %k~ith (ampvto/acter. Abisorpti~ cel iAprotrude Intoi lunien of gut and remain unclee h~r bacteria. Bactri with characteris~tic spiral ,hapc Iarrl~s adhere to region', hclteeriahsorpti~e cell,,. Bar = i km.

\11 ik(iI )WOI % (

4N I

I h ttr I .i~ ~111, . 1rifr/' I(M I i IO\ IC IrMa~ Ci fr %N Il Iuiiiiii~iI sillItAC ot lf \ els N.jnh ai earrc

- '4 C.,N,1-( -,. Fdo tol 0Ih"-4-h M cls N ee te 2 ni nthto . Ine £, enavnci f1cei em- no\ls~ o ie

11Ii111 4k lidra S tsj acptial tuhs acls N en~fe 2 mi iiira aar m i caion. net 'Ts uiae c t ofa bac ina h final e ndo c of I(X n [, mni

Ruitri112itresin n'r,\ kth5vd usesosofapoi trJimed/ an contpurf iue d toifi int frs a oo em eatrFcalce 0 \ctu mcli Conro loots rhinocuateidig. -2.0h kg.il cutue inatstal oops2 h.Spcie f or ina4Csoluion an caig electron

hraethe test ine 70a retrne ki ( kliite ado.4 al caz kg acernthe mioralscpy 2.5M ad Slur. resp ectoc \kerc ucrolu tleclr s~i 111*\lI inals \kee tostd kior at luras.tXI prdretorme\ Thle stil 56)) 45 Tn sd u calacu oide (reagent Irde osmiu 1(trx)iidC '(idto atern ~i iclation ;lopt sepu.l throg anna r cuthenraie laprotii dccodr ate. is r ethatilsers. madecimns frIlal \lune embedded. oEan si spt tion .h ican Hoopst easuring on Ani semad easin intle Ep ae at2 'Thic 7..stec ntilfxtion PPer Ser diseed and I''mo~cicbu

hro The intesinle NJ returned ino the lhopsa %cr itemared the adI / micrscop 1T1' n 11diu bMrec(ser ee grae souion 'hn)

perri dish, drained. and inverted over a glass roid. sectiomns %~ere stained sw th I '4; Lranvlacetate and Re\ nold's lead

.1 . 11 ,t''l l r '1! 1 1 l - l l

a'1J11(C l t aId ( . 1S i i k ~ l'11 T I lk 1 \ I'1 , il \

I l c l Il c m&'' ' , 1 \ 1 1 1 k l .IIIII1

I WIl Vj I Ik k, 1 Cd C l 'i\ ,I . I)II%" ,kit f ( e 'h l 11 J ' lik1 k ,1\- 1111 1I' 10 1L10 1 1

I Ik I. I iI

IN ao..i t ed~i~ \k Ii iic11 nic rl "CII r II Iphi [it IdCN id I'l l o I nh )[ofit II I 111 .C L Ik Ci ninI IchI icli'iiCCN I Lrticic rc (.1 Ii, I 9X S.Y(L sttN c I

ci l 11 9 SI . ji'icxc c \ c tt IN ia\ nfl hi' asNofciaited xk I iI c nic roii\.NICpitipcIICN Ofiih thriin hint 111,x he dUC io artcl\

haci:criatl nitAkc s\ Nstem of the hoti. Iu III, MCIhjxron1 Is Lon"INicil x ti he uptaike (it lloInl "IxI~i i ioicra OrIiaisiN Ilk

M~ I iiN C ( )x cn Ct dl. 1986) 111 cittrax! 10 a rcp0rI bo, ( )x criand Jo~nes 1974) and anoticr h\ lLInjliriurat 1 i986) dcx inu

Z4' hc Uipiakco IWGi Ilk; h\1 celis. [hc pNCLIdotdii kc uptakc (11bactcria h\ (ic MI cell xaN IMC arcex N III iir NILIii\

Iisof interest that lit iini\ arc iact baCicria taken kip Ilk(he Mi kcCIINO u also 11rix orcltilisllNh CIt Ctraiisict red NilliLli

I tliCOhisi, to ll]Clk11W- i\ iiipiiiii 101iiiJCN Fli'iC nIIIIIIICrNo~f ti '_alINsills pliacedi Iii int lo ps iia\ he rcsponsihIc lor tir

driiiatic. uptake seenI n thiN N(Lnd\ Rapid Upiake of bacteriacold leadl to transienit bacicriinia h\ ox crx liclniiiii noiitiial

CA clearance ichaniis. [h !cillilln i hc seenI %k heliel 'a N'iil.I

pliciitinicriiti Ini 11iiii1IiuoCoiiipr-lllNCLI hostN cOldii dC011Uill itir

U opporiti~14c jiilcctillsN Ilic of)iiit il hi i\iipliai I aidblood CiiCiLIdt~r\ sxxcm miii iakes thiN a likci\ possihiliix

thcem to ihc uridcrix inl'- folljic This process hcax NiilaritijeN

hian Ca t- 19 1 rroh Ii duI ircrx Ir r II itlN I nr

cachn adria E~vipin 'mn chc. z i) a nNCCI1 ~~~ ~ ~ Oke iii ll. 1961 Itultc xxo tri rthtcAmdL s Radii ncx Rcsarcphenonatuc seen i

NI_:AkW CCI16 attcre lop iru re-nen nchhose ekeas ociuTt)I Ibllvc 1usri

in crtan acaNxx ilcica rigrii a sor~ixc cithiia ccix n oes iurterton cIIicann k olr re ai to~ iie iiiic

xx ~ dr~rirI nd u ii CTI:NI.XX it TENIt baacrter cc ia ec 1111.1. andre~ Htraspote [111.-017.1-1 M Cells a[o h hi INh

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