DTIC ADA501904: The Toxicity of Soman in the African Green Monkey (Chlorocebus aethiops)

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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
The toxicity of soman in the African green monkey (Chlorocebus aethiops)

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6. AUTHOR(S) 5d. PROJECT NUMBER
Despain, KE, McDonough, JH, McMonagle, JD, McGinley, MJ, Evans, J
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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT
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AUNSD A ArDmDyR MESeSd(iEcaSl) Research Institute of Aberdeen Proving Ground, MD
Chemical Defense 21010-5400 USAMRICD-P06-015
ATTN: MCMR-CDC-V
3100 Ricketts Point Road

9. SPONSORING / MONITORING AGENCY NAME(S ) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
US Army Medical Research Institute of Aberdeen Proving Ground, MD
CChheemmiiccaall DDeeffeennssee 21010-5400
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13. SUPPLEMENTARY NOTES
Published in Toxicology Mechanisms and Methods, 17(5), 255-264, 2007.

14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Acetylcholinesterase, African green monkey, cardiac troponin I, ECG, EEG, intramuscular, soman, telemetry, LD50, nonhuman primate,
rhesus macaque
16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON
OF ABSTRACT OF PAGES Kenneth E. Despain
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ToxicologyMechanismsandMethods, 17:255-264,2007 informa
Copyright©InformaHealthcare healthcare
ISSN: 1537-6516print; 1537-6524online
001: 10.1080/15376510600972733
The Toxicity of Soman in the African Green
Mon!<ey (Chlorocebus aethiops)
Kenneth E. Despain
Comparative Medicine Division, ABSTRACT This studydetermines soman toxicityin African green monkeys
United States Army Medical (Chlorocebus aethiops) and is the first step in exploring the suitability of this
Research Instituteof Chemical species as a model for nerve agent studies. Male African green monkeys were
Defense, 3J00 Ricketts Point
surgicallyimplantedwithtelemetrydevicestomonitorelectroencephalographic
Road, Aberdeen, Proving
(EEG) and electrocardiographic (ECG) activity. Blood was taken at various
Ground, Maryland 2J010-5400
times to measurewhole blood acetylcholinesterase (AChE) activityandcardiac
John H. McDonough, Joseph
troponin I (cTnl). Blood AChE activity relative to baseline was 0.0% to 2.5%
D. McMonagle, Maura
6 h after soman exposure and recovered to 31.9% to 72.0% by 30 days after
J. McGinley, and
.Jackie Evans exposure. The 6 h postexposure cTnl levels varied from 0.64 to 6.55 ng/mL,
Pharmacology Branch, United suggestingcardiac damage. Somanwas prepared in saline to aconcentrationof
States Army Medical Research 100{Lg/mL. Usinganup-down design for small samples, subjectswere exposed
Instituteof Chemical Defense,
to 5.01, 6.31, or 7.94 {Lg/kg soman 1M. The first subjectwas given 5.01 {Lg/kg
3J00 Ricketts Point Road,
soman 1M and survived. Three subjects received 6.31 {Lg/kg soman 1M and
Aberdeen, Proving Ground,
survived. Three subjects received 7.94 {Lglkg soman 1M and died within 25
Maryland 21010-5400
min, 26min, or6h. Inall subjects, toxicsigns ofmuscle fasciculation, tremors,
Received 17July2006;
accepted23August2006. chewing, and profuse salivation developed within 2 to 7 min. Tonic-clonic
Theopinionsorassertionscontained motorconvulsions and EEG seizure began between 2 and 18 min after tremor
herein aretheprivateviewsofthe
onset. The 48 h 1M LD50 ofsoman in saline in the African green monkey
authorsandarenottobeconstruedas
officialorasreflectingtheviewsof was calculated to be 7.15 {Lg/kg. The signs and speed ofsoman intoxication
theDepartmentoftheArmyorthe
in: African green monkeys were consistent with those described in rhesus,
DepartmentofDefense.Theauthors
would liketorecognizethe cynomolguscynomolgus, and baboons.
outstandingsupportofSGTJason
McKainandSPCChristinaA.Dansie
KEYWORDS Acetylcholinesterase;AfricanGreen Monkey;CardiacTroponinI;ECG;EEG;
andtheotherveterinarytechnicians
oftheComparativeMedicineDivision Intramuscular; LD50; Nonhuman Primate; Rhesus Macaque; Soman; Telemetry
whoassistedwiththiswork.
Inconductingtheresearchdescribed
inthisreport,theinvestigators INTRODUCTION
adheredtotheGuidefortheCareand
UseofLaboratoryAnimalsbythe Phylogenetically,nonhumanprimates(NHPs)aretheclosestanimalstoman.
InstituteofLaboratoryAnimal
Inbiomedicalresearch,theyareconsideredtobetheanimalthatphysiologically
Resources, NationalResearchCouncil,
inaccordancewiththestipulations most closely approximates how a ~rug or toxin would act in man (Miller
mandatedforanAAALAC
1967; Dixon 1976; Krasovskii 1976). The rhesus monkey (Macaca mulatta)
Internationalaccreditedfacility.
has traditionally served as the NHP research species ofchoice to assess nerve
Addresscorrespondenceto KennethE.
Despain, ComparativeMedicine agent toxicity and the effectiveness ofvarious medical countermeasures. The
Division, UnitedStatesArmyMedical
"choice" of the rhesus monkey seems to be a case of historical default. In
Research InstituteofChemical
Defense, 3100RickettsPointRoad, the late 1940 s, animal research involving toxicology ofnerve agents and the
Aberdeen ProvingGround,
developmentofmedicalcountermeasureswasfirstinitiatedintheUnitedStates
Maryland21010-5400
E-mail: [email protected] andvariousalliedcountries.Atthattime,rhesus monkeyswere readilyavailable
255
and widely used for biomedical research. Since then, monkeys are similarto rhesus in anatomy, physiology,
researchinthenerveagentarenaappearstohavesimply hematology, blood chemistry, and social organization
followed this trend. Some investigators examined com (Fairbanks 2002). They are considerably less aggressive
parative intraspecies (e. g., monkeyvs. dog) differences than rhesus, and well-trained personnel can perform
in toxic mechanisms and/or response to therapies repeated blood sampling from superficial veins. Wild
(DeCandole et al. 1953; DeCandole and McPhail caught, Caribbean origin, African green monkeys are
1957; Johnson et al. 1958), while others used rhesus available from a variety ofsources for around 30% of
to characterize the effects ofmedical countermeasures the costofarhesus monkeyand, mostimportant, they
against specific aspects of nerve agent toxicity (Lipp do not carry Cercopithecineherpesvirus 1.
1968, 1972, 1973; Lipp and Dola 1978; Dirnhu,ber African green monkeys are used in biomedical
et al. 1979). research for behavior, AIDS, diabetes, genetics, infec
India, the major foreign source of rhesus mon tious disease, neurobiology, and cell biology studies
keys, stopped exporting these animals for biomedical (Fairbanks 2002). However, there are no previous
research purposes in the late 1970s. Subsequently, toxicological or pharmacological research studies with
increasing demand for these animals, coupled with nerve agents or any of the standard medical coun
limited domestic breeding sources, greatly decreased termeasures using African green monkeys. Before an
availabilitywhile substantially increasing cost. In addi informed decision can be made about the suitability
tion, rhesus monkeys pose a serious health hazard to of the African green monkey for future research, it
research and husbandrypersonnel due to the potential is necessary to determine the comparability of data
for transmission ofCercopithecineherpesvirus 1(monkey obtainedwith the African green monkey to historical
B virus), which has exceptional virulence in humans data already available for other NHPs including the
(Artenstein et al. 1991; Davenport et al. 1994; Holmes rhesus monkey. This studywas designed to determine
et al. 1990). Protecting personnel from this virus the toxicity ofthe nerve agent soman in African green
requires additional personal protective equipment and monkeys for comparison to otherprimate species. The
special medical monitoring, all ofwhich increase the 1MLD50 dose ofthis agentwas to beestablished along
overall husbandry and research costs of using these with a description ofthe time course and severity of
animals. For these reasons, there is a need to find physiologicalsignsofintoxication.Anemphasizedgoal
an alternative monkey species to evaluate nerve agent ofthis work was to use as few animals as possible for
toxicityand medicalcountermeasures againstchemical this determination.
warfare nerve agents. Two other NHP species, the
cynomolgusmonkey(Macacafascicularis) (Carpentieret MATERIALS AND METHODS
al. 2001; Krummer et al. 2002; Lenz et al. 2005; Lalle
Animals
mentetal. 1997, 1998, 1999,2000,2002;vonBredowet
al. 1991)and the common marmoset(Callithrixjacchus) All animalswere housedandcaredforinaccordance
(D'Mello and Scott 1986; Muggleton et al. 2003; with the Guide for the Care and Use of Laboratory
Wetherelland French 1991;vanderSchans etal. 2003; Animals(NationalResearchCouncil1996).Sevenadult
van Helden et al. 1992, 2003, 2004a, 2004b; Busker male, wild-caught African green monkeys (Chlorocebus
et al. 1996; Philippens et al. 2000), have been used aethiops) from the island ofSt. Kitts, weighing 4.5 to
in this regard. Both ofthese species have drawbacks. 7.0 kg, served as subjects. Animals were housed indi
The cynomolgus monkey also carries Cercopithecine vidually in 4.3-square-foot stainless steel squeeze-back
herpesvirus1, thus posingahealth riskequivalentto the cages with built-in perches. Theywere fed commercial
rhesus monkey. The marmoset is substantially smaller certified primate ration by Harlan/Teklad (15%) (W),
(body weight 250-450 g), which hinders the ability to fresh fruit, and tap water ad libitum. Animal rooms
take repeated blood samples ofsignificant quantity. were maintained at 21 ± 2°C, relative humidity of
TheAfricangreenmonkey(Chlorocebusaethiops) may 50% ± 10%, and a 12-h light (0600-1800):12-h dark
be an idealreplacementforthe rhesus monkey. African (1800-0600) cycle with no twilight. The U.S. Army
green monkeys are old-world monkeys that grow to MedicalResearchInstituteofChemicalDefenseisfully
about 60% the size ofa rhesus and are from the same accredited by the Association for the Assessment and
subfamily as baboons and macaques. African green AccreditationofLaboratoryAnimalCareInternational.
K. E. Despainetal. 256
The Institutional Animal Care and Use Committee side served as one EEG channel, the temporal and
(IACUC) approved the research presented here. occipital screws on the right side served as a second
EEG channel, and the frontal and central screws on
Soman the right side served as a third EEG channel when a
four-channel transmitterwas used (three animals). The
Soman was obtained from the Edgewood Chemical
frontal andcentralscrews on the leftside served as one
Biological Center, Aberdeen Proving Ground, MD. It
EEG channel and the temporal and occipital screws
was assayed to be >98% pure by nuclear magnetic
on the right side served as a second EEG channel
resonance analysis. It was diluted in saline to a
when the three-channel transmitter was used (three
concentrationof1.95 mg/mLandmaintainedasfrozen
animals). The leads were trimmed to length, a small
stock at -80DC; purity was further verified by gas
('"'-'0.5 cm) part ofthe silastic covering ofthe lead was
chromatographyto be 98.7%. From this frozen stocka
removed, and the bare lead wire was wrapped around
single 1-mLvialwas slowthawedanddilutionsmade to
the shaft of the screw; the screw was then turned
prepare multiple vials containing 1mL of100 f-ig/mL
into the skull, anchoring the wire in place. The screw
soman in physiological saline. These 1-mL vials were
head and wire leads were covered with dental acrylic
thenfrozen at-80DC and singlevials were removed for
and the incisions closed using absorbable suture. Lead
the dosingofeach animal.
II electrocardiographic (ECG) capability was created
when electrodes from the transmitter were tunneled
Surgery
subcutaneously and placed beneath the overlaying
Sixofthesevenanimalswereimplantedwithcortical musculature using 2-0 Prolene to anchor the wire
electroencephalographic (EEG) leads and a telemetry electrodetothebodywall.Thenegativeleadwasplaced
device (animal V5342 was not implanted). Animals in the upper right chest quadrant near the heart base,
were fasted overnight prior to surgery. Each animal and the positive electrode was placed near the heart
wasanesthetizedinitiallywithketamine1M(10mg/kg), apex in the lower left chest quadrant. Muscle and
then intubated. Further anesthesia was maintained fascia were closed in layers using a simple interrupted
using isoflurane (0.8%-2% with nitrous oxide and pattern with absorbable suture; skin was closed with
oxygen in a 2:1 ratio). The animal was then placed in an intradermal continuous pattern using absorbable
aKopf(Turlingua, CA) stereotaxic frame andprepared suture. All skinincisionswere reinforcedwithVetbond
for sterile surgery. Approximately 15 min before the tissue adhesive (3M, St. Paul, MN). Each animal
first incision was made, the antibiotic cephazolin was receivedasystemicantibiotic1M(40,000units/kgpeni
administered IV (25 mg/kg). Approximately 10 min cillin G benzathine) and an analgesic 1M (buprenor
before the first incision, a mixture of epinephrine phine, 0.01 mg/kg) postoperatively. Approximately 4
(1:1000, 0.3 mL), lidocaine (2%, 0.5 mL), and sterile to 6 weeks passed between surgery and nerve agent
physiologic saline (0.9%, 2.2 mL) was infused around exposure.
incision sites to provide local anesthesia and promote
hemostasis. A midline incision was made in the scalp.
Burr holes were drilled (bilaterally for four-channel
EEG and ECG Recording
telemetry device) in the s~ull over the frontal and
central sites, right temporal andright occipital cortices, EEG recordings were obtained by telemetry from
and a frontal midlin.e for ground, and then a stainless freely moving animals in their home cages using DSI
steel screw was inserted into each hole. An incision DataquestART (version 2.3) software and a computer
was then made 5 to 8 cm below the left scapular monitor to display the signals. At least three baseline
region ofthe back and a subcutaneous pouch created recording sessions, 18 to 24 h in duration, were
to accept the biopotential transmitter device (Models obtainedforeachanimalpriorto nerveagentexposure.
TLlOM4-D70-EEEE, four channel or TLlO M3-D70 Recordings were obtained continuouslyduring and for
EEE, three channel; DataSciencesInternational [DSI], 48 h after agent exposure. In surviving animals, 24-h
St. Paul, MN). The leads from the transmitter were EEG records were made on days 3, 10, 15, 30, 45,
tunneled subcutaneously from the subscapular region 60, 75, and 90 throughout the 3-month survival time
to the skull. The frontal and central screws on the left before euthanasia. ECG recordings were obtained by
257 Soman Toxicityin Monkey
telemetry from freely moving animals in their home acetylthiocholine iodide as asubstrate. Ten microliters
cages using DSI Dataquest ART (version 2.3) software of whole blood was pipetted into a labeled 0.5-mL
and a computer monitor to display the signals. ECG tube containing 190 ILL of sterile water and mixed
recordings were used on the day of soman exposure thoroughly. These tubes were then frozen to -80aC
to assess the clinical condition ofsubjects during their until thawed for AChE measurement. A SpectraMax
cholinergic crisis. Plus 384 microtiter spectrophotometer (Sunnyvale,
CA) and a PC with associated SoftMax controller
Blood Collection software were used to analyze samples.
Blood was drawn from the saphenous vein uS,ing
25-gauge needles affixed to 1-mL syringes precoated Troponin Assay
with 1000 USP units/1 mL sodium heparin. The
Plasma was obtained from all animals before and
venipuncture site was clipped and swabbed with 70%
after soman exposure. After 10 ILL of whole blood
isopropyl alcohol, and ~1.0mLofbloodwas taken for
was taken for theAChEassaypreviouslydescribed, the
each sample.
blood samples were centrifuged for 10 min at 14,000
rpm. The plasma was transferred to a 3-mL Nalgene
screw-top vial and frozen to -80aC until thawed for
Nerve Agent Exposure
cTnI measurement. A TOSOH AlA 600 Automated
Onthedayofexposure,abloodsamplewasobtained Immunoassay Analyzer (TOSOH MEDICS, INC.,
to determine pre-exposure blood acetylcholinesterase South San Francisco, CA) was used to determine cTnI
(AChE) and cardiac troponin I (cTnI) baseline values. plasma levels. A single measure of cTnI was made
The soman vial was slow thawed and the appropriate for each plasma sample. The AlA-PACK cTnI second
dose drawn up in a syringe within a safety hood, then generation is a two-site immunoenzymometric assay.
placed on ice within a sealed container for transport This assay is performed entirely in plastic test cups
ofthe syringe to the animal quarters. The animal was containing lyophilized magnetic beads coated with
then injected with the predetermined dose of soman anti-cTnI mouse monoclonal antibody and mouse
(0.100 mg/mLin saline) in the calfmuscle while being monoclonal antibody to cTnI that is conjugated to
briefly restrained in a squeeze-cage. The injection site bovinealkalinephosphatasewith0.1%sodiumazideas
waswipedusingagauzespongewettedwitha1%bleach apreservative.ThecTnIpresentinthetestsamplebinds
solution followed by wiping with a water-dampened with the monoclonal antibodyimmobilized on amag
sponge.Thesyringeandgauzewipeswereplacedinfull netic bead and enzyme-labeled monoclonal antibody
strength bleach solutions for decontamination. Three in the antibody immunoassay test cup. The magnetic
soman doses were used in the study: 5.01 ILg/kg (N = beads are washed to remove unbound enzyme-labeled
1),6.31 ILg/kg (N = 3), and 7.94 ILg/kg (N = 3). EEG monoclonal antibody and are then incubated with a
recording began within 1min ofinjection and clinical fluorogenic substrate,4-methylumbelliferylphosphate.
observations were performed continually for at least 4 The amount ofenzyme-labeled monoclonal antibody
h following exposure. Additional blood samples were bound to the bead is directly proportional to the cTnI
obtained to measure AChE at 6 h, 3 days, 10 days, concentration in the test sample. A standard curve
and 30 days after exposure in survivors; the 6-h blood is constructed, and unknown sample concentrations
samples were also assayed for cTnI. are calculated using this curve (TOSOH Medics,
2001).
Acetylcholinesterase Assay
Experimental Design
Blood samples were transferred to microfuge tubes
containing30ILLoflOOOUSPunitsll mLsodiumhep An up-down design for small samples was used
arin to prevent clotting. Blood AChE determination (Dixon and Massey 1981). The starting dose was
was performed using a microtiter plate modification 5.01 ILg/kg (loglO = 0.70), which is roughly halfway
of the WRAIR method (Feaster 2000) that utilizes betweenthe reportedsomanLD50 for rhesus monkeys
K. E. Despainetal. 258
(7.4{ig/kg1M;Adamsetal. 1976)andthatofcynomol Each challenge dose elicited a distinct progression
gus monkeys (3.7 {ig/kg 1M; Adams 1990). Based on and duration oftoxic signs. The animal (V564) dosed
the toxic response ofa particular test animal (alive vs. with 5.01 {ig/kg ofsoman had the longest latency for
dead at 48 h), the next soman dose was increased or seizureonset,andtheseizuresterminatedafteralmost1
decreased in a0.1 10glO unitincrementforthe nexttest h. Shortly after termination ofthe seizures, the animal
animal. slowly regained consciousness, although tremor, fasci
culations, and salivation were still evident for 4 to 8 h
as coordination and normal behaviorreturned. Within
RESULTS
2 days, this animal appeared normal. Three animals
Clinical Toxicity to Soman (V471,V576,V584)were dosedwith6.31 {ig/kgsoman
1M.These animals experienced seizure durations of2.5
Soman doses of 5.01 {ig/kg, 6.31 {ig/kg, and
to 3.5 h, and it was 2 to 8 h after the seizure ended
7.94{ig/kgelicited severe signs ofnerve agentintoxica
before evidence of consciousness (response to sound
tion. Within minutes ofsoman injection, the animals
or touch, voluntary movement) returned. One animal
developedchewingand/orfacialautomatisms.Thiswas
(V471) initially appeared to recover, but displayed at
immediatelyfollowed bymild and intermittenttremor
least three spontaneous seizures (2- to 4-min duration)
in the limbs, which shortly progressed to strong and
several days after the intoxication. This animal then
continuous tremor in the whole body accompanied
failed to eat or drink and was given subcutaneous
by facial grimacing. This phase was soon followed
fluids. Evenwith these measures his physicalcondition
by uncoordinated thrashing movements that rapidly
deteriorated to the point where a decision was made,
progressed to tonic-clonic convulsions, EEG seizures,
6 days postexposure, to euthanize him for humane
loss of posture, and unresponsiveness to external
reasons. In contrast, the other two animals displayed
stimuli. A profuse, thick, ropy salivation developed
uncoordinated behavior that slowly resolved over 1
andpersisted throughout theperiod ofseizure activity.
to 3 days postexposure, after which they appeared to
EEG seizure activity and motor convulsions were
fully recover. Three animals (V331, V361, V5342) were
prominentfeatures ofintoxication.Table 1summarizes
intoxicated with the 7.94-{ig/kg dose ofsoman. After
the times for seizure onset and their duration. Figure
the initial progression of signs, two animals (V361,
1provides an example ofthe EEG record ofa seizure.
V5342) developed chaotic (Cheyne-Stokes) respiratory
There was a notable cycling of seizure/convulsive
efforts 10to 15minafterexposure;cyanosisdeveloped,
activity; it would primarily be characterized as clonic
seizure/convulsive activity rapidly declined, and then
with intermittent intense episodes of tonic activity.
periodsofapneadeveloped,accompaniedbydepressed
Afterthefirst 15to30minofseizure,therewas notable
heart rates. Both animals died about 25 min after
waxing andwaning ofseizure/convulsive activity, with
exposure. The third animal (V331) displayed seizures
periods of2 to 4 min quiescence in epileptiform EEG
for 1h 50 min. The seizure spontaneouslyterminated,
activity and convulsive movements between episodes
after which the animal continued to salivate profusely
of seizure/convulsive movements. This waxing and
over the next 4 h; there was a slow, steady decline in
waning would become more prominent as the seizure
body temperature and heart rate until the animal died
duration grewlonger (>1h).
6h afterexposure.
TABLE 1 Onsetoftremors,seizureonset,seizureduration,andoutcome
Subject Soman dose Tremoronset Seizur~onset Seizure duration Outcome
V564 5.01 ~g/kg 7min 17min29sec 57 min Lived 90 days
V471 6.31 ~g/kg 3min 6min 38sec 3h23 min Euthanized 6days
postexposure
V576 6.31 ~g/kg Ei min 14min 11 sec 2h 54min Lived 90days
V584 6.31 ~g/kg 2min 4min 46sec 2h29 min Lived 90days
V331 7.94~g/kg 2min 7min 5sec 1h 50 min Died 6h
V361 7.94~g/kg 2min 2min 21 sec 21 min Died 26 min
V5342 7.94~g/kg 3min 5min 13sec 18min Died 25 min
259 Soman Toxicityin Monkey
...
It ...+-' p • It.... : ,
:
,.,
1.'1) t'i§ 2:0 2.~ s.b
~:
,,'n s.b
...
~~II~'I.r:"II.J.tll-:- ;_,,~:r.q.~.'.\'~/llIjlj~llil'h~UIII.I'~.-:
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~",~\YI.~NW~I1II!~~~I~~.'_'J~!!~
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elmeotEEG
Mlnuit.
FIGURE 1 Acontinuous75-minrecordofEEGfollowingtheadministrationof5.01 ltg/kgsomantosubjectV564.Eachx-axislineis5
minandy-axisismillivoltamplitude;theleftfrontal-centralscrewsaretheEEGleadsrecorded.Somanwasgivenat9:04AM;therecord
starts 1min later at9:05 AM. Approximately 14 min afterthe start ofthe recording, there is anotable and sustained increase in EEG
amplitudethatculminates inseizureonsetat17.5min after injection.Theseizurecontinues uninterrupted atvery highamplitudesfor
almost5min andthen shiftsinto periodsofwaxing and waning ofdifferentdurationsforthe next52 min,where, atthe endofaburst
ofrapid high-amplitudespiking,theseizureabruptlyendsanddoesnotreoccurfortherestofthe24-h record. By10min afterseizure
terminationtheanimalbehaviorallydemonstratedsignsofalertness.
K. E. Despainetal. 260
TABLE 2 Percentacetylcholinesterase(AChE)activityaftersomanexposurerelativetobaseline
Subject Soman dose 6 hAChE activity 3 daysAChE activity 10daysAChE activity 30daysAChE activity
V564 5.01 fLg/kg 1M 1.5% 3.5% 6.6% 72.0%
V471 6.31 fLg/kg 1M Undetectableactivity 0.3% Died Died
V576 6.31 fLg/kg 1M Undetectable activity 1.1% 22.5% 31.9%
V584 6.31 fLg/k9 1M 2.5% 2.0% 7.7% 45.2%
V331 7.94fL9/kg 1M 0.3% Died Died Died
Blood Acetylcholinesterase and and peripheral sites causing a buildup of the neuro
Plasma Cardiac Troponin I Levels transmitter acetylcholine and sustained activation of
acetylcholine receptors. Excess acetylcholine initiates
Relative to baseline values, whole blood AChE
rapid progression ofmiosis, hypersecretions, muscular
activitywasseverelydepressedbysomanexposure,with
fasciculation, tremors, seizures, convulsions, and death
nosignificantdifferencebetweendoses.However,there
in laboratory rodents, NHPs, and man (Sidell 1997;
was a notable recovery ofenzyme activity by 30 days
Tryphonas and Clement 1996; Baze 1993).
after exposure. Table 2 summarizes the blood AChE
Therateofsomanintoxicationandobservedclinical
activity relative to baseline for subjects surviving 6h, 3
signs in African green monkeys were consistent with
days, 10 days, and 30 days after exposure to soman.
descriptions in the literature for the rhesus monkey,
Blood samples taken 6 h after exposure for the
cynomolgus monkey, and baboon (Adams et al. 1976;
three monkeys that received the 6.31-fLg/kg dose of
Adams 1990; Anzueto et al. 1986). As Lipp (1968)
soman had elevated cTnl levels. Table 3 summarizes
had indicated, electrographic tonic-clonic seizures and
the baseline and 6-hcTnllevels forthese threeanimals.
motorconvulsionswere prominentaspects ofthe toxic
symptomatology ofsoman intoxication in all animals
at the doses studied, although it must be emphasized
Animal Dose Sequencing
that only one animal was exposed at the lowest
and 48-h Survival
dose.
Table 4 displays the sequence in which the animals Cardiacdamage following somanexposure has been
were exposed, the response, and the detailed calcula reported in rhesus and cynomolgus monkeys, as well
tions recommended by Dixon and Massey (1981) to as in baboons (Britt et al. 2000; Baze 1993; Anzueto
estimatethe1MLD50ofsoman. Notethatthevariance et al. 1986). The data in Table 3, obtained from
(a) inthiscalculationisassumedtobeequivalenttothe single samples tested with no replicates, demonstrate
step size(theincrementbetweendoses). Basedonthese
calculations, the estimated 48-h 1M LD50 ofsoman is TABLE 4 Summaryofanimalresponsesand LD50
7.15 fLg/kg (6.28-8.13 fLg/kg = ± 1 SEM). calculationsfortheup-downmethod
Dose Log10
(fLg/kg) Dose Response (AnimalTattoo)
DISCUSSION
7.94 0.9 X(V331) X(V361) X(V5342)
6.31 0.8 0(V471) 0(V576) O(V584)
Soman is a lethal organophosphorus nerve agent
5.01 0.7 0(VS64)
thatirreversiblybinds to acetylcholinesterase atcentral
o
=48-hsurvival; X=fatality.
L050 = Xi +(K x d).
TABLE 3 AfricangreencTnlplasmalevelsatbaselineand L050 =0.9+(-0.458 x 0.1).
postexposuretosoman L050 =0.9-0.0458.
L050= 0.8542(1og10)
6h Postexposure L050=7.15fL9/kg(6.28-8.13 f-Lg/kg =± 1SEM).
Subject Soman dose BaselinecTnl cTnl Xi = lastdosetested= 0.9(loglO).
K= Oixon&Masseytable 19.2= -0.458.
V471 6.31 fL9/k9 1M 0.00 ng/mL 0.99 ng/mL d= intervalbetweentestdoses= 0.1(10910).
V576 6.31 fL9/k9 1M 0.00 ng/mL 6.55 ng/mL Nt= total numberofsubjectstested= 7.
V584 6.31 fL9/k9 1M 0.00 ng/mL 0.64ng/mL N= samplesize= 6.
0" (variance)=d;SE=0.560".
261 Soman ToxicityinMonkey
TABLE 5 Soman LD50valuesinnonhuman primatespecies
Species LDso (SurvivalTimes) Route Diluent Reference
Rhesus 12.9ltg/kg' SC Notstated Fukuyama and Askwich 1963
Rhesus 9.5 ltg/kg' 1M Saline Lipp 1968
Rhesus 7.4ltg/kg (24 h) 6.65 ltg/kg 1M PEG-DH 0 Adams etal. 1976
2
(5 day)
Rhesus 12.3Itg/kg* SC Notstated Dirnhuberetal. 1979
Rhesus 7.3 ltg/kg (48 h) 1M Saline Olson etal. 1997
Cynomolgus 3.77 ltg/kg (24 h) 1M PEG-DH 0 Adams 1990
2
Baboon 6.65 ltg/kg (24 h) IV Saline Anzueto etal. 1986
'Survivaltimes notstated.
that cTnl increases to clinically significant levels in include the African green monkey. A highly sensitive,
the African green monkey after soman exposure, very specific biochemical marker for soman-induced
indicatingthatcardiacdamagehasoccurred.According cardiac injury in the African green monkey would be
to assay specifications, within 4 to 8h following acute a valuable tool in nerve agent toxicity and medical
myocardial infarction, cTnl will increase above levels countermeasurestudies. Thereis apossibilitythatcTnl
that indicate that myocardial infarction has occurred isjustsuchabiomarker,andanalyticalvalidationofthe
(TOSOH Medics 2001). The 6-h time point selected cTnl assayfor the African green monkeyis warranted.
for cTnl determination is the median of4 to 8 hand Table 5 displ\lYs reported LD50 ofsoman in three
we hypothesized that cTnl levels would be elevated by large NHP species. In the present study, the 48-h 1M
this time ifcardiac damage had occurred. LD50 of soman in African green monkeys was 7.15
NoneoftheAfricangreenmonkeysinthisstudyhad fLg/kg. This dose is almost identical to the 24-h LD50
gross orhistopathologic cardiac lesions at 90 days after s reported for soman in rhesus monkeys and baboons
soman exposure. However, 48 to 72 h is the optimal by other investigators (Adams et al. 1976; Olson et al.
time to assess histological changes associated with 1997; Anzueto et al. 1986). However, the 3.77-fLg/kg
myocardial degeneration and necrosis (Schoen 2005). 48-h 1M LD50 of soman in cynomolgus monkeys
By90days afterinjury, small,multifocalcardiaclesions . reported by Adams (1990) appears to be excessively
characteristicofsomantoxicityarehealed,leavinglittle low based on the consistent LD50 values obtained in
orno evidence that theyeverexisted. the other NHP species. Also, from the literature,it
Three isoforms oftroponin I identified are skeletal appears thatSCinjections ofagentin this speciesresult
slow twitch, skeletal fast twitch, and cardiac. These in a higher LD50 value than do 1M injections. The
threeareproductsofdifferentgeneswithuniqueamino 24- or 48-h survival period for LD50 determinations
acid sequences (Adams et al. 1993). Of the three, withnerveagentsissomewhatarbitrary.Althoughmost
cTnl is found exclusively in cardiac tissue. Though deaths from nerve agent intoxication occurwithin the
the three isoforms share some homologous structure, first hours following exposure, deaths can continue for
cTnl has a sequence of31 amino acid residues at the several days even after apparent recovery, as was seen
amino terminus ofthe molecule, which renders cTnl with subject V471 in this study. This has beenseen in
antigenically distinct from the other two isoforms of other NHP studies with nerve agents (von Bredow et
troponin I, as well as from troponin C and troponin al. 1991; Koplovitz et al. 1992; Murphy et al. 1993) as
T (Katrukha 2003). Monoclonal antibodies developed wellas rodentstudies (McDonough etal. 1989;Shihet
to react with the epitope in this unique portion ofthe al. 1990). Thus, the LD50 estimates at shorter survival
cTnl molecule have made possible the development timeswillbehigherthan thosebasedonlongersurvival
ofin vitro diagnostic assays with great specificity for end-points.
cTn!. Monoclonal antibodies against human cTnlwill The primary advantage of the up-down method
cross-react with cTnl of the rabbit, baboon, rhesus is that it concentrates testing near the mean, which
macaque, andotherspecies (Cummins and Perry 1978; increases the accuracy with which the mean can be
Walker 2006). Our findings support the premise that estimated (Dixon and Massey 1981). This advantage of
cTnl is well conserved across a variety of species to concentrating testing at the medial lethal dose proves
K. E. Despainetal. 262
m: '7
valuable when determining the LD50 for toxic agents death by anticholinesterase poisoning. Br. J. Pharmaco/. 8:466
such as soman. It is noteworthy that the LD50 value 475.
DeCandole, C. A, and McPhail, M. K. 1957. Sarin and paraoxon
establishedinthisstudyusingtheup-downmethodwas
antagonism indifferentspecies. Can. J. Biochem. 35:1071-1083.
accomplishedwithlessthanhalfthenumberofanimals Dirnhuber, P., French, M.c., Green, D. M., Leadbeater, L.,andStratton,
J.A 1979.Theprotectionofprimatesagainstsomanpoisoningby
used in previous studies utilizing more traditional
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our results are comparable to and ofthe same level of Dixon, R. L. 1976. Problems in extrapolatingtoxicity datafor laboratory
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precision as results obtained using traditional probit
Dixon, W J., and Massey, EJ. 1981. Introduction toStatisticalAnalysis.
design, it is highly recommended that this up-down NewYork: McGraw-HilI.
D'Mello, G. D., and Scott, E. A M. 1986. Toxicity of sarin and GD
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in marmosets (Callithrix jacchus). Technical Paper 458 Chemical
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