Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202- 4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To) 2007 Open Literature 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER The toxicity of soman in the African green monkey (Chlorocebus aethiops) 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Despain, KE, McDonough, JH, McMonagle, JD, McGinley, MJ, Evans, J 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER 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 ATTN: MCMR-CDZ-I 11. SPONSOR/MONITOR’S REPORT 3100 Ricketts Point Road NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution unlimited 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 a. REPORT b. ABSTRACT c. THIS PAGE UNLIMITED 10 19b. TELEPHONE NUMBER (include area UNLIMITED UNLIMITED UNLIMITED code) 719-526-1218 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 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'~.-: ~ ~S 3] U ~O U ~O •.b ~",~\YI.~NW~I1II!~~~I~~.'_'J~!!~ 2] U 3] 3.S ~ ~,~.~,",r~1~~~ '~II-'" :'" •• 2~ u ~ 3.S Q U 1~ U 2] U 3~ U ~ s.b 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 pretreatment with pyridostigmine. J. Pharm. Pharmaco/. 31:295 probitmethods(Adamsetal. 1976;Adams 1990). Since 299. our results are comparable to and ofthe same level of Dixon, R. L. 1976. Problems in extrapolatingtoxicity datafor laboratory animalstoman. Environ. Health Perspect. 13:43-50. 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 approachbeused in all such future studies thatinvolve in marmosets (Callithrix jacchus). Technical Paper 458 Chemical NHP species as a means of reducing the number of DefenseEstablishment, Porton Down, United Kingdom. animals used to obtainvalid results. Fairbanks, L. 2002. Alternative old world primate models for non-AIDS research; African green monkeys (vervets). In Rhesus Monkey BasedontoxicresponseandLD50value, theAfrican DemandsinBiomedicalResearch;aWorkshopReport. ed. National greenmonkeyrespondstothelethaleffectsofthenerve AcademyofSciences. Bethesda, MD: Research ResourcesInforma tionCenter. agentsomaninanalmostidenticalfashiontotherhesus c.. Feaster, S. R., Gordon, R. K., Doctor, B. P., Clark, C. Maxwell, D. M., monkeyandappears tobeanacceptableNHPmodelto and Lenz, D. E. 2000. Unprocessed whole blood cholinesterase assess mechanisms ofnerve agent toxicity and medical levels: WRAIR protocol development and validation. USAMRMC BioscienceReview. 3:1099-1107. countermeasures. Fukuyama, G. S., and Askwich, W E. 1963. Unpublished data cited in Adamsetal. (1976)andDirnhuberetal. (1979) Holmes, G. P., Hilliard, J. K., Klontz, K. c., Rupert, A H., Schindler, REFERENCES C. M., Parish, E., Griffin, D. G., Ward, G. S., Bernstein, N. D., and Bean, T. W 1990. B virus (Herpesvirus simiae) infection in Adams,1. E. III, Bodor, G. S., Davila-Roman,V G., Delmez,1. A, Apple, humans:epidemiologicinvestigationofacluster.Ann.Intern. Med. ES., Fred, S., Ladenson, J. H., and Jaffe, A S. 1993. Myocardial 112:833-839. injury/infarction: cardiac troponin I: amarkerwith high specificity Johnson, R. P., Gold, A J., and Freedman, G. 1958. Comparative forcardiacinjury. Circulation88(1):101-106. lung-airway resistance and cardiovascular effects in dogs and Adams, N. L. 1990. The intramuscular lethality of soman (GD) in monkeys following parathion and sarin intoxication. Amer. J. the cynomolgus monkey. USAMRICD-TR-90-06 US Army Medical Physiol. 192:581-584. ResearchInstituteofChemicalDefense,AberdeenProvingGround, Katrukha, A 2003. Antibody selection strategies in cardiac troponin MD,ADA224887. assays. In CardiacMarkers, Second edition. ed. A H.B. WU. New Adams, N. L., von Bredow, J., and de Vera, H. V 1976. Intramuscular Jersey: HumanaPress. lethalityofGD (soman) in the rhesus monkey. EdgewoodArsenal Koplovitz, I., Gresham, V. c., Dochterman, L. W, Kaminskis, A, TechnicalReportEB-TR-7603 Aberdeen Proving Ground, MD, AD and Stewart, J. R. 1992. Evaluation of the toxicity, pathology, A026821. and treatment of cyclohexylmethylphosphonofluoridate (CMPF) Anzueto, A, Berdine, G. G., Moore, G. T., Gleiser, c., Johnson, D., poisoning in rhesusmonkeys.ArchToxicol. 66:622-628. White,C.D.,andJohanson,W.G.1986.Pathophysiologyofsoman Krasovskii, G. N. 1976. Extrapolation ofexperimentaldatafrom animals intoxication inprimates. Toxicol. Appl. Pharmaco/. 86:56-68. toman. Environ. HealthPerspect. 13:51-58. Artenstein, A W, Hicks, C. B., Goodwin, B. S., and Hilliard, J. K. 1991. Krummer, S., Thiermann, H., Worek, E, and Eyer, P. 2002. Equipotent Human infection with Bvirus following a needlestick injury. Rev. cholinesterase reactivation in vitro by the nerve agent antidotes Infect. Dis. 13:288-291. HI 6 dichloride and HI 6 dimethanesulfonate. Arch. Toxico/. Baze,W. B. 1993. Soman-induced morphological changes: an overview 76(10):589-595. inthenonhuman primate.J. Appl. Toxico/. 13:173-177. Lallement,G., Demoncheaux,J. P, Foquin, A, Baubichon, D.,Galonnier, Britt, 1. 0., Martin, J. L., Okerberg, C. V, and Dick, E. J. 2000. M., Clarencon,D.,and Dorandeu, F. 2002. Subchronicadministra Histopathologic changes in the brain, heart, and skeletal muscle tionofpyridostigmineorhuperzinetoprimates:comparedefficacy of rhesus macaques, ten days after exposure to soman (an againstsomantoxicity.DrugChem. Toxico/. 25(3):309-320. organophosphorusnerveagent). Compo Med. 50:133-139. Lallement, G., Clarencon, D., Masqueliez, c., Baubichon, D., Galonnier, Busker, R. W, Zijlstra, 1. J., Philippens, I. H., Groen, B., and Melchers, M.,Burckhart,M.,Peoc'h,M.,andMestries,1.C. 1998.Nerveagent B. P. 1996. Comparison ofthe efficacyofsingle or repeated HI-6 poisoninginprimates:antilethal,anti-epilepticandneuroprotective treatmentfollowingsomanpoisoninginguineapigsandmarmoset effectsofGK-11.Arch. Toxico/. 72:84-92. monkeys. Toxicology112:183-194. Lallement, G., Clarencon, D., Galonnier, M., Baubichon, D., Burckhart, Carpentier, P., Foquin, A, Dorandeu, F., and Lallement, G. 2001. Delta ,M. F., and Peoc'h, M. 1999. Acute soman poisoning in primates activity as an early indicator for soman-induced brain damage: a neitherpretreatednorreceiving immediatetherapy:valueofgacy review. Neurotoxico/ogy22(3):299-315. c1idine(GK-11)indelayed medicalsupport.Arch. Toxico/. 73:115 Cummins, P., and Perry, S. V 1978.Troponin Ifrom human skeletaland 122. cardiacmuscles. Biochem. J. 171:251-259. Lallement, G., Mestries, J. c., Privat, A, Brochier, G., Baubichon, D., Davenport, D. S., Johnson, D. R., Holmes, G. P, Jewett, D. A, Ross, S. Carpentier,P.,Kamenka,1.M.,Sentenac-Roumanou,H.,Burckhart, c.. and Hilliard,J. K. 1994. Diagnosisand managementofhuman M. E, and Peoc'h, M. 1997. GK-11: promising additional neuro Bvirus(Herpesvirussimiae)infectionsin Michigan. Gin. Infect. Dis. protective therapy for organophosphate poisoning. NeuroToxico/. 19:33--41. 18:851-856. DeCandole,C.A, Douglas,W.W, Evans,C. L.,Spencer, K. E.,Torrance, Lallement, G., Renault, F., Baubichon, D., Peoc'h, M., Burckhart, M. F., R. W,and Wilson, K. M. 1953. The failure of respiration in Galonnier, M., Clarencon, D., and Jourdil, N. 2000. Compared 263 Soman Toxicityin Monkey