DTIC ADA436057: The Potential Neurotoxic Effects of Low-Dose Sarin Exposure in a Guinea Pig Model

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by  Defense Technical Information Center

THE POTENTIAL NEUROTOXIC EFFECTS OF LOW - DOSE SARIN EXPOSURE
IN A GUINEA PIG MODEL

Melinda R. Roberson, PhD, Michelle B. Schmidt, SSG Mary D. Gonzales and
John H. McDonough, Jr., PhD
Pharmacology Division, U.S. Army Medical Research Institute of Ch emical Defense,
Aberdeen Proving Ground, MD 21010 USA

ABSTRACT

This study is assessing the effects in guinea pigs of repeated low - dose exposure to the nerve agent
sarin. Preliminary results suggest no effects of either repeated 0.2 or 0.4 X LD50 sa rin exposure (co m-
pared with s a line) on body weight or temperature, general physical signs, flinch threshold or activity
level, or on EEG activity. In contrast, RBC cholinesterase levels dropped to 20% of baseline following
the tenth e x posure in the 0.4 gr oup. Since this study is ongoing, data from receptor binding and brain
cholinesteraasssheaai nyds s topatholsaotrgbieyel cilon lgl ecaatnneaddl yiznbmaefeandl yd,u enbcye d
the dramatic changes in cholinesterse a c .ytivit

INTRODUCTION

A great dea l of research has been conducted to study the single acute effects of chemical warfare
nerve agents (CWNA) and how to protect against the acute toxic effects. Consequently, the s e quence of
events following a single, seizure - eliciting exposure to organophos phorus (OP) CWNA has been rel a-
tivelwye lclh aracterizeHdo .w everm,u clhe sisks n owanb outth eef fectosrf e peateldo dwo seex posure
to OP nerve agents, and there has been concern that such exp o sure may have contributed to the adverse
healtehf fectrse por GvWubtuetalyenthrtfD dcee o u er era tnaseti.fhon fefpte ryoco ftl so ndlgooeswde
chemiecxaplo bsnetuettoreauth ed niee, dsrr e er stptaohntede ntaial d verhseea lctohn sequenscouefcs h
exposure and to dete r mine what level of exposure may produce ad verse effects.
The experiments detailed here are intended to furnish initial data that should address a number of
fubfraTrootttshfeirnhhhieoss geaesessreso nrts emaui d ue rcen d cosgihn e.cs u rreenxtaelmlyie ncet r o-
physiological, behavioral, biochemic al, neurochemical, and histop a thological parameters in an animal
model that utilizes repeated exposure to low levels of the nerve agent sarin.
neuntruhbomesebeh teTeoraunhhfovrd ea ueeirvs peeeli efs oooaf wfrte aecldt s - leexavp eolts ou re
yteirav fo OP agents such as DFP or paraoxon (see Russell and Overstreet, 51 1987 for a r e view), and
some limited studies with nerve agents. Previous studies of repeated administration of the nerve agent
soman shows that such treatment produces transient, but revers ible, changes in regional brain chol in -
1
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
00 JAN 2002 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
The Potential Neurotoxic Effects Of Low-Dose Sarin Exposure In A
5b. GRANT NUMBER
Guinea Pig Model
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Pharmacology Division, U.S. Army Medical Research Institute of REPORT NUMBER
Chemical Defense, Aberdeen Proving Ground, MD 21010 USA
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
This article is from ADA409494 Proceedings of the 2001 ECBC Scientific Conference on Chemical and
Biological Defense Research, 6-8 March , Marriott’s Hunt Valley Inn, Hunt Valley, MD., The original
document contains color images.
14. ABSTRACT
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
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a. REPORT b. ABSTRACT c. THIS PAGE UU 7
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Prescribed by ANSI Std Z39-18
esterase (ChE) activity and regional brain muscarinic receptor (mAChR) numbers. 9 ,3 During the time of
repeated exposure the animals can display altered behavior and may develop a tolerance to the agent as
the exposu res co n tinue. 41 ,21 In addition, it has been reported that repeated low level exposures to sarin
mayp roducep ermanenta lterationsi nb raine lectroencephalographic( EEG)s pectrumt hatf aro utlastt he
period of exposure. 2 Because of this persistent change inE EG,t hereh aveb eenc ontinuingc oncernst hat
exposure to low doses of nerve agents can also produce neural lesions in brain such as those seen a f ter
exposure to high doses of nerve agent that produce prolonged seizures. 8 ,7
In the present study, guine a pigs, previously instrumented to record EEG activity, were exposed
daily (5 days/wk for two weeks) to two doses (0.2 X LD50 and 0.4 X LD50) of sarin. Measures of red
teawmbcepotiebdigrtEoyfvhahEd littrGyiCt,ue n(hy rs(cnE,ehRh, ,oB c lCedb)lsl l o od ociceptiona)n,d
generaalc tivitlye velwse rdee termineddu rintghe ex posurpeh asawese lha2lrs s 31,,0 3 ,0o 1,r0 d 0a ys
following e x posure in different groups.
Tthweo - napewegerexoervepfsnieko etow sl daue sbrc eetec pextaedphour oifspiss erou odr e vides
sufficient time for ChE to be driven to a low, stable level. The nerve agent, s a rin, and the doses (0.2 and
0.4 X LD50) were selected to allow replication and expansion on a dat a base of low - dose work begun at
USAMRICD. 1dTpohaserestsei, c utlhaerl y nopatrdaadnpovlboohdhesls 0yuiroee.scgs ,4iehe o e lr o g -i
cal effects such as weight loss or lethality. The current LD50 has its basis in protection studies histor -i
cally conducted in guinea pigs at USA M RICD.

MATERIALS AND METHODS

General Methods: Guinea pigs (Final N = 180) are anesthetized with isoflurane and stereotaxically
im planted with stainless steel cortical screw electrodes. 11 ,01 Following a one - week recovery period and
initihaaln dlidnag(yT Msu,b ) a,s eliEn(Eme3Gi 0 n /dayb)a,s elibnle oCohabdEne ,dh aviordbaailts a e-
ing gathered on two pre - exposure days (W, Th). Animals are then i n jected daily, s.c., 5 days/wk for 2
wkwsi tsha linoesr a ri(n0 .2o,0r .L x4D 50L;D 54=ì02 g /kg). exeodpafOaoc nysh u brtoeed,my p er a-
ture( pre - a ndp o st - exposuraebn)od wd eyi gahmrtee a sureadtn,ahd ne i maalrse monitorfEeaoEdrG c-
tivity (power spectral analysis, broken into five EEG bands) for 15 minutes to e s hsil b aaytliad pre -
exposure baseline. After each injection the animals are monitored for E EG activity for 1 hour and are
assessed for general signs of sarin exposure, including eyelid closure, facial tremor, fasciculation, writ h-
ing, vocalization, circling, biting, the ease of handling, lacrimation and salivation. Following EEG r e-
cording, anima ls are assessed for change on measures of nociception (flinch thres h old/foot shock) and
aChE RBCa noaflf ysoserdil asey costn de rda wni sB loogadec nte irvailt y. c terminatthieo n After tivity.
etxhopefo spuhraess eep,a rgaratoneoui fpme asavr laesl ua tfEeaobEdnreG d h a v 10i,c3o h,ra aanltg es
30 and 100 days. In addition, at each of these times (plus at 2 hrs post exp o sure), groups of animals are
euthanized( 75m g/kg,i .p.,p entobarbital)a ndt ra n scardiallpye rfuseTdh .be r aiann hde aratr ree moved ,
and regional brain ChE activity, regional brain receptor B and K ,a ndb rainn europathologya reb eing
xam d
de[termi ned. 3 ]H -Mtob indmiAnCgh R for usedb einPgi riesn zepine rece p tors, 5 [and 3 ]H - CGP - 396i5s3
1
glfubuotesraie mndag t (eNNrMgDiAc - Dmethyl - asparrteacbteiepn)td oirn g. 61 Sep a hbrearaanatrdiet n
evalhuiasfttoiorosp ntasattn ihoeunofcmil hbnoneagagirs iiwq sccniueaacteosrllhasusr u r,tmie ee ndt d-
ing hematoxylin and eosin (H & E) and glial fibrillary acidic protein (GFAP). 6 Apo - tag will b e used to
identify potentially apoptotic ne u rons. 31
Nociceptive (flinch) thresholds are determined by the up - and - down procedure. 4 The animal is
placed in a test chamber (16 cm L; 11 cm W; 13 cm H) with a stainless steel grid floor through which
gniyrav intensities of electric shock can be delivered. After a 1 - min habituation period, single shock
pulses (0.5 sec) are delivered at 15 - sec intervals. Shock intensities are available from 0.05 to 4.0 mA in
20 steps arranged log a .yllacimhtir hcnilF si gniebd efined as any visual withdrawal reaction in response
2
shtpoorc eks ent a tahdea potAfan t iroense paavoccanhcrs toiieorebns. ddeth i weonincaglsknt li di t oyn ,
“up -presdeoenfott refdroetmrurhi s ene idsn iasgsmm pallfmeloeds rto hwond” a ishock of tion ntensities
premiodfp Tohien t s ereidaeucsrh.i ng - expboassmueerlaei ne s fsrtpoaomri ttnhiten g uarssee mrevnetss
which the shock intensities will be varied for each animal.
General activity is measured for 30 minutes in a 40 cm X 40 cm X 30 cm clear plexiglas cha m ber
utilizingag rido fp hotob eamsH. o rizontala ndv erticala ctivity( numbero fb eamb reaks)i sm easuredi n
10 - min segments along with total activity for the 30 - msiens sHiaob ni.t uatibioesni dnegf intaedhsde e-
enilc ni ytivitca sa acnuf tion of time during the session 41 (results not shown).
Animal Care and Handling : Research was conducted in compliance with the Animal Welfare Act
and other Federal statutes and regulations relating to animals and experiments involving animals and a d-
here s to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research
Council, 1996. The facility where this research was conducted is fully accredited by the Association for
Assessment and Accreditation of Lab o ratory Animal Ca re International.

TABLE 1. General Experimental Scheme.

Implant
edortcelE enilesaB cinorhCerusopxE ts oP - erusopxE

llAsS s y a2 d M / sk e e2w - F 2 srh 3 01 03 001syad

(EE G, (EEG, )ygolo t,smieHhco r,ureoNiv a,hGeEBE( Animal #
B e )roivah )roivaheB latotbuS

Saline 6, 6 6, 6 6, 6 6, 6 6, 6 60

Sarin: 0.2 LD50 6, 6 6, 6 6, 6 6, 6 6, 6 60

S arin: 0.4 LD50 6, 6 6, 6 6, 6 6, 6 6, 6 60
180 Total

Two groups of 6 animals at each time point: six are being tested for flinch threshold and six for activity
level. Animals from both behavioral groups are being used for ChE and rece ptor assays, and neurop a-
thological evalu a tion.

RESULTS

Tphree sernetp orretp resenitnst erriems utlohotfins sg oisntgu dSyi.nw cidetoc hasoi enn d itioanl,l
animalasrt er eatetdhs ea mdeu rintghe ex posurpeh aset,h eraers eu fficienatn imaltso deotearnmyi ne b-
vious trends that may have developed during this phase of the study (See fi g ure legends for Ns).
Durdtiahnpiegol syt - exposuErrEeeG c ordipnegr ih(or1dt) h,e wreer e no obsesr avsraoiibfgnl n es
e x posure on behavioral and sensory indices: eyelid closure, writhing, vocalization, circling, biting, and
the ease of handling, lacrimation and salivation. For further discussion of the results, see Figures 1 - 7:

3
egnahC thgieW ydoB

600

ENILAS
500 LD50X 0.2

LD50X 0.4

400

300

Weight Gain (g)
200

100

0
enilesaB Da1y Da2y Da3y Da4y Da5y Da6y Da7y Da8y Da9y 10Day Days3 01syaD 03syaD sy a0D01

syaD erusopxE erusopxE tsaL retfa syaD

Figure 1. e gtn hayghdiCoeBW :dino was There f 2.0 rehtie dna enilas neewteb )g( niag thgiew ydob ni ecneref
or 0.4 X LD50 sarin animals over the 2 - kpexeew o erus doirep ro pu ot01 0stysaodp - .erusopxe sN( =02 - 23
animals for all three groups from initial the baseline day throughout the e x e r ,u,dsrooeipvreewp otha 3 syad
after the last exp o sure, Ns = 16 - ;91 ta 01 ,syad sN =11 - ;41 ta 03 ,syad sN = ;9 dna ta 001 ,syad sN =2 - 3.)

erutarepmeT ydoB

37.9
37.8
ENILAS
LD50X 0.2
37.7 LD50X 0.4
37.6

37.5

37.4
Temperature (°C) 37.3

37.2

37.1

73

enilesaB Da1y Da2y Da3y Da4y Da5y Da6y Da7y Da8y Da9y 10Day Days3 01syaD 03syaD sy a0D01

erusopxE tsoP erusopxE tsaL retfA syaD

Figure 2. erutarep myedToB :and saline between (ºC) temperature body in difference significant no was There
either 0.2 or 0.4 X LD50 sarin animals. Te m erutarep nwohs no erusopxe syad1 - 01 saw nekat 1 ruohtsop -

injection. All temperature mea serus ni siht noitrop fo eht yduts erew nekat gnisu a latcer eborp ISY(ehT r-

.,e)teir hsFes gtN e yie(=msrdemaaooWosfBm g ure 1.)

4
delevar Tecnatsi DlatoT

0052

enilaS

0002 LD50X 0.2
LD50X 0.4

0051

Disance (cm) 0001

005

0
enilesaB ya1D ya2D ya3D ya4D ya5D ya6D ya7D ya8D ya9D y0a1D sy a3D sy a0D1 sy a0D3 sy a0D01
syaD erusopxE erusopxE tsaL retfa syaD

Figure 3. )l eyvteiLvi tdceAl(e veacrnTa tlsaitDoT neaos oftdocef fos enaewre h:T c d e ,ts laayet tvsaievlit

function of distance traveled (cm). The large jump in acti v yti de v lrn eei svslepebleu loarothratg 3 syad

retfa eht tsal erusopxe si ylekil eud ot eht dnekeew kaerb dna eht ecnesba fo eht noitcejni .gnildnah ddA i-

cominutes, 15 only fort esatcitnigv)i ty to (pricohra mber EEG the in present are antiimoanlasl ly, m pa o tder
1 hr, 15 min on injection days. (Ns = same as for Flinch Threshold (see Figure 5), except for the 100 - yad
time point in which (at this point in the study) Ns = 1 for the saline and 0.4 LD50 groups, and N = 0 for the
0.2 LD50 group.)

Change in Nociception (Flinch Threshold)

57.0

ENILAS
07.0 0 5 2DX.L0
0 5 4DX.L0

56.0

06.0
mA

55.0

05.0

54.0
enilesaB 1 yaD 2 yaD 3 yaD 4 yaD 5 yaD 6 yaD 7 yaD 8 yaD 9 yaD 01 yaD syaD 3 syaD 01 syaD 03 syaD 001

Exposure Days Days after Last Exposure

Figure 4. Change in Nociception (Flinch Threshold) : er etlshcuaTefw fg ffnonoein inerasaeosm dno a
measuroenf o ciceptio(nf lincthh resholdS)h .o wanr rea fwl incthh reshold(sm Af)o prr e - doire perusopxe

,enilpexseab o s y1eardus - 10 (measured 1 hr post - ,)no iddtenctae cjsenyli aerdsetf a ee.rhdutosiorpexpe

sN( =9 - 61 rof lla eer hstpuorg r oefn ihl geeusroaurbshotp xyead ; 0,1revewo hta 3 syad retfa eht tsale x-

,eru ssoNp =6 - 11; at 10 da s,=N6sy - ;9 ta 03 ,syad sN =3 - 6; and at 100 days, Ns = 1 - 2.)
5
PoPwreer-TTorteaalt ment PowerTotal Post-Treatment

0042 ENILAS 0042
05D L X2.0 ENILAS
0002 05D L X4.0 0002 0055DD LL XX24..00

0061 0061
2mv)m 2v)mm
0021 0021

EEG Power ( 008 EEG Power ( 008

004 004

0 ileseaBn 1 erusopxE 3 erusopxE 5 erusopxE 6 erusopxE 8 erusopxE 01 erusopxE 0 nilesaBe 1 erusopxE 3 erusopxE 5 erusopxE 6 erusopxE 8 erusopxE 01 erusopxE

gnilpmaS GEE rof stnioP emiT detceleS gnilpma SGE Ero fstnio Pemi TdetceleS

Figure 5. Pre - Treatment Total EEG Power : There Figure 6. tsoP - Treatment Total EEG Power : There
saw on tnacifingis tceffe fo rehtie eht 2.0 ro4 .0 saw onis g tnacifin tceffe fo rehtie eht 2.0 ro4 .0
05DL niras esod no latot GEE rewopvì( 2 ), when 05DL niras esod no latot GEE rewopvì( 2 ), when
pre - erusopxe doirep enilesab GEE sawoc m pared the pre - erusopxe doirep enilesab GEE s erusaem

with the 10 - yad erusopxe doirep pre - injection EEG erewoc m derap htiw erusopxe doirep yliad tsop -

r e cordings ( F = 0.60, P > 0.83). (For Figures 3 inj noitce EEG r e cordings ( F = 1.035, P > 0.43).
12,6 12,6
and 4, N = 11 (saline); N = 12 (0.2 LD50), N = 13 (On both pre - and post - treatment EEG, analysis of

(0.4 LD50.) eht evifdni ividual bands (spectral analysis: ä, è, á,
â , â ) osla detluser ni on tnacifingis tceffe fo niras
1 2
esod nodni i lau d di)nv.arbewop

leveL esaretsenilohC CBR

061

ENILAS
041 05D LX2.0

05D LX4.0
021

001

08

06
Percent of Baseline
04

02

0
enilesaB erusop xtEsoP erusop xtEsoP erusop xtEsoP sya D3 sya D01 sya D03 sya D001
1 5 01 erusop xtEs arLet fsayaD

gnilpmaS EhC CBR rof stnioP emiT detceleS

Figure 7. leve Lesaretseniloh CCBR a EhC CBR ,sngis GEE ro laroivaheb fo ecnesba eht ot tsartnoc nI : c ytivit
dropped to less than 20% of baseline following the tenth exposure in the 0.4 LD50 sarin an imals, and to less

tha n 40% of baseline in the 0.2 LD50 animals. Both agent groups showed a steady increase in ChE acti v yti

following the exposure period, with 0.2 animals returning to baseline and 0.4 an imals remaining just below

enilesab ta 001 syad retfa eht tsale x .erusop
6
CONCLUSIONS

It is important to reiterate that these results are preliminary, since the study is ongoing. While it is
informative to gain an idea of the direction the results may be taking, drawing definitive concl u sions at
thpiosi wnotu pblrede matFuurr et.h etrrh,ee c eptboirn dianbngrd a CiahncE t iviatsys aaytnshhd ie s t o-
pathologicaaln alyseasrc eu rrentlbye incga rrieodu tc;o nsequentltyh osdea tsa coinsufficiently are ets m-
plete to be pr e sented here.
However, during the pre - exposure period baseline and exposure day pre - and post - injection mea s-
uretNassh'r,uese f ficientllayrn( gu~aem2op b0onae)f rr a metaetlrolss op we c u havwfea rS,o lat ion. e
obbeshenalrovove iwiefoondffrda e ainclc t ao tfi on - dsoaesrxeip ndots losahcesuanaehaertdt esse t , d-
ule used here. However, the drop of RBC ChE activity to b e low 20% of baseline in the 0.4 LD50 sarin
group, and to below 40% of baseline in the 0.2 LD50 group, represents a dramatic contrast. How this
change in ChE activity will affect receptor binding, brain ChE activity and neuropathology is not known.
Neverthelestsdh,r4eta o02b onop%0a df %s eliCnahecE t ivisitusyf ficiestnuotg getshtaa t lt eratiionn s
intracellular param e ters -- either biochemical or histopathological -- will be observed in the sarin - exposed
animals, at least transiently (at the earlier time points) and perhaps persi s tentl(ya 3ta0 n 1d0 d0a ypso st -
exposure). When the study is complete, it may be possible to connect these suggested neurochemical
and/orn europathologicala lterationsw iths omeo ft hea dverseh ealthe ffectsr eportedb yG ulfW arv ete r-
ans.

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