EDCEWOOD ARSENALSPECIAL PUBLICATION

EASP 11004

SUTILIZATION OF [3 2 p] SOMAN FOR MEASUREMENTOF ACETYLCHOLINESTERASE IN BRAIN TISSUES

by

Norman C. Thomas

Joseph H. Fleisher

Larrel W. Harris

April 1972

NATIONAL TECHNICAL \• ,INFORMATION SERVICE U

DEPARTMENT 3F THE ARMY[NONO ARSENAL

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EiArmul, MlqW 21010

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Distribution Statement

This documett. aay be further distributed by :he holder only with specific priorapproval of the Commanding Officer, Edgewood Arsenal, ATTN: SMUEA-TS-R, EdgewoodArsenal, Maryland 21010.

The findings in this report are not to be construed as an official Department of theArmy posit'on unless so designated by other authorized documents.

Disposition

Destroy this report when no longer needed. Do not return it to the originator.

... ... ..

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UNCLASSIFIEDSecurity Ossaificatin-

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CO, Edgewood Arsenal UNCLASSIFIEDATT`N SMUEA..BL-RBB 12 fO.

Edgewood Atsenal, Maryland 21010 NA

UMLIZATION OF (3 P) SOMAN FOR MEASUREMENT OF ACETYLCIHOLINESTERASE INBRAIN TISSUES

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bound to the tissucc as mrethyl 132p, phcsphonate. The methyiphosphonate for 100'ý inhibition was takenequivalent to ¶Jie catalytic sites. Turnover numbers calculated from the ratio of acetylcholine hydrolysis tocatalytic sitet. approachcd vaiucs yielded by eel acctykcholinesterasc.

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5I

EDGEWOOD ARSENAL SPECIAL PUBLICATION

EASP 1100-6

uTI IZATION OF 13 2p] SOMAN FOR MEASUREMENTOF ACETYLCHOLINESTERASE IN BRAIN TISSUES

by

Norman C ThomasJoseph H. Fleisher

Larrel W. Harris

Medical Research Division

April 19722

Approved for public release. disoribution unlimited.

Task I W662710AD2502

DEPARTMENT OF THE ARMYEDGEWOOD ARSENALBiomedical Laboratory

Edgewood Arsenal. Maryland 21010

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FOREWORD A

The work described in this report was authorized under Task W.6627 1OAD2502, MedicalDefen.se Against Chemical Agents. Prophylaxis and Therapy for Lethal Agents. This work wasstarted in November 1967 and completed in Julv 1969. The experimental data are contained innotebook MN-2135.

In conducting the research described in this report, the investigators adhered to the"'Guide for Laboratory Animal Facilities and Care" as promulgated by the Committee on the Guidefor Laboratory Animal Resources. National Academy of Sciences-National Research Council.

IAThis report is reprinted from Biochem. Biophys. Acta 235, 542-547 (197 ) by permission

of the copyright owner. The letter of permission to use the copyrighted ,raterial is on file in theLegal Office. Edgewood Arsetial. Maryland 21010. Reproduction of the -xpyrighted material inwhole or in part is prohibited except with permission of the copyright owner: however, DDC andthe National Technical Information Service are authorized to 'eproduce th:s document for UnitedStates Government purpose.,

Iq7

DIGEST

Aceiylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) activity in brain tissues of thedog was measured before end after inhibition with 3 2P-labeled pinacolyl methylposphonofluoridate(soman). A high correlation was obtained between uninhibited enzyme activity and soman-derivedradiophosphorus bound to the tissues as methyl 13 2P1 phosphonate. The methylphosphonate fo'100% irhibition was taken equivalent to the catalytic sites. Turnover numbers calculated from t&%ratio of acetylcholine hydrolysis to catalytic sites approached values yielded by cclacetylcholinesterase.

A

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Repdnted frorn

Biochatnics et Biophysica Acts m

Elscv.ier Publishing Comrpany, Arnurtrdarn Printed in The Netherlands

BBA ReportKIBHA 61225Utilization of 1 P1 somran for rne-auremnwrt of acetyllcholinesterews inbrain tissues

NORMAN C. ThIOMIAS, 30SEP11 11. FLEISHJER and LARREL W, HARRISA%'ý'Me&d&icalSinccs bcp~r'ment-WMdical Research L boatoirs Edgeuvod Arsenal. Ml. 11010

(Rccived April 6th. 1971)

SUMMARY

Acetylchotincsterase (acetylcholine hydrolase. EC 3. 1. 1.77) activity in braintissues of the dog was measured before ar.d after inhibition with 32 P-labeled pinacolvimethylphosphonofluoridate (soman). A high correlation %us obtained betweenuninhibited enzyme activity and sonian-derived radiophosphorus bound to the t.issuesas methyl [32pj phosphonate. The niethyilphosphonate for 100% inhibition was taker.equivalent to the catalyvtic sites. Turnover numnbers calculated from the ratio o-acetylcholine hydrolysis to catalytic sites approached values yielded ble eel acewv:

- cineterase

It is well established that ishibition of acetvlcholincsterase (acetylcholinehydrolase, EC 3.1.1.7) by organophosphorais compounds in 7irro takes place througit

phoshork~ton o th en m- 3. Howvecr, no correlation has been observed in estrobetween the radiophosphottis bound to mammalian Itissijes following injection of -Plabeled diisoptopylphosphoroiluoridate (DFP) or isoptopyl niethylphosphopnofluoridate -(sarnh) and the normad acelylc-holkncstcrase content of tuch tissucs*di. The lack o'correlation obscrved int theset stul-ies must be regarded as inconclusiv since no distinctionwas made between phosphorus speciflwally bound to acetylehoilinesterase and that bound

nonspcifcalyto thr rot~n?'and many isus The distinction and measurernen:of specific binding of oho-phones in relation to ihbiOn f aCetvlcholineterase Ir rnlreand in virr, has been under study i ou r iaboira-Unn~~ Wc havc shown tt1Z3 irn rats oven

aP~ati. Cat orton f rait aewliloinete,5ewhich could itc reactivated in Yv.rytby 2.pynidinturni aldoximne metisochloride ajprom~mated the prcemntag of phospho.rU-released as isoppyl itythylpbospliowac. The amount of phosphorus retairied by theenzyme after incubation with 2-pyridiniun. aldoxime metlchloride was present entirely,

bb-khf. 8*ippikys At". 235 (1971) 542-S47

BBA REKORT 543

as niethyl[ 2PI phosphonate and paralleled the percentage of enzyme not reactivated("aged" enzyme). The close correlation between the enzymatic and radioactivity measure-ments suggested that the method distinguished photsphorus bound to acetylcholinettcraseI ~ from that bound to other site?~.

linacolyl methylphosphosiofluoridate (sontan) like DFP and sarin phosphonylatesboth acetyleholinesterase mnd non-specific sites'. It diffirs from DFP and sarin inproducing an inhibited acetylcholinesterase which is only slightly reactiviated by oximes".In explanation. Fleisher and Harris' showed that the acetylehoinesterzse phosrhonylatedby t3*Pl sortan in ritro undergoes a very rapid decrease in reactivatability ir. parallel withWloss of a pinacolyl group (dealkylatiord. Rapi-d dealkylation of somari-inactivated etythru-cyte acetylcholincsterase and eel acetylckolinetterase in rigrr has also been shown byCout et a!. and Michel et al ' respectively. In agreement with the low acetylcholin-esterase. acvtv of homogenates of rat live; anti lune compared~ to that fromt brain24 thesoman-derived phosphoryl residue in liver and lcng tissue showed little dealkyiation compzredito that in brain tisbue of rat!, given 111sonan8s.

These findings suggested the possibility that the niethylphosphonate content ofa tissue might ve related to the numbet of acerylc-holinrtcerase sites following inactivation.by suntan. Expetriments designed to test this possibility in brain tissue 5re descrbe in thisreport.

Anaestbietized dogs werc perfused with hcparinized 0.9%. saline until the brain wasvirtually free of blood. The brain was removed after sacri ice; and the caudate nucleus.thalamus, and portions of the medulla. hippociinptis, cerebral1 and Ccreblm-lar co.te,. were ýexcised, wvisased anti used for the following studies:

(Aji Rate of locs of a'ractivarabifity r*aging"!, in vitro. Homogenates of -each tissue(20T wlvj were p.-epored in 0.,11 M borate buffer at rill 8.8 and O?. The preparations wereincubated with 2- -O 10 simai. (final concentration- fo 30 min at e?. 30-90% inhibitionwith minimal aging resulted. The trxture was imnicuately equilibrated to 370 -Reactivatabil.ity by monoisonit~rusaccrone was r..-osred before, and at suitable time intervals aftcr, adjustingthe pH to 7.4 tue initiate aging as desccribeud previously'. The M. te of loss of reactivalabiliny bynionoisonitros-,ameone was first order with a haif tirti of (6.04 t 0.58 min (P1= 0-9S) forIS preparations (3 for each brain tissue). This -ate doentdfersgiiatl rmiareported earlier by us for dog erythrocyte acetyicholmnesterase inhlibited~bv soinan' is-and it istaken as approximating th.- rate of dealkylation'.

01) Ratkio of -ivdrol)vsis o1 acetvlcholtne to act'-3mtwloiein i~fmogenairsof dog brnd tasisse This study was roerf;'muned in otde~r that turnover numbers for acetyi-,3.methylehoiine in dog brain tissues could be converted to 3cetylCholinc (Table 1).

Acetyicholinesterase activity for-both substrates was measured radionictricall-according to Salakotos, eta)"16. The rmethd is based upon the adsorption of unhydrolyzed

substrates as their 1 IJTJ acid choline esters on Amnberlite CG-l 20 resin suspended indioxaiie. The, supernatant solution containing the product of hydrolysi%. the free 1-14C)I.

arid- is counted in a liquid scintillation spectromreter. Details concerning the preparation ofsubstralms the resin, and the scintillation cocktail (fluor) are given b~y Siakoto* e A 1.~'

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UHA REPORT 545

I & homognates of the brain tissues were prepared in water. The cerebral cortexhomogenate was further diluted to 4%(w!v), that from caudate nucleus to 0.2.... and theremaining tissue hovmog,'nates to 1% with water. For assay, 0.1 ml of homog nate and0.1 nl of 0.1 M sudium phosphate buffer at pH 7.4 containing 0.3 M raCl and 1% LubrolWX (I C. I.OrganicS Inc.. Stamford. Conn.) were put in each of two centrifuge iubes.100pl of 3- 0- M 1.[ Cj acetylchohne iodide (2.5 mC/manolei was added to the firstsample and the same volume o - 3 M [ t.'C] acetyl-3-methylcholme "4.16 mCimmole)to the second sample of each homogenate. The contents were mixed iminediately andincubated for 5 mini at 37*. Preliminary studies showed substrate hydrolysis to bx- linearwith time under these conditions. S ml of dioxanc-resin suspension wee then added toeach tube The mixture was then made to 10 ml with dioxane. mixed. centrifuged, mid 5 mlof the supernatata was removed and counted. The reading were corrected t:.i, non-eniyviatic hydrolysis of the labeled substrate with conroi solutions contai.i.ng 0.1, mi! ofbuffer medium and 0.1 nil of water in place of tissue. The readings (counts!mii) werereferred to a standard curve relating radioactivity (countesmin) to pmoies - !0-3 of thelabeled substrate. "Tne total hydrolysis of substrate per g of tissue per min was calcul'ittdfrom the following formula:

It•mes I 3 10 M I 1000mgmlmi .5 mi- mg ot homogenate used

The values so obtain-ed for the hydrolysis of l14 C1 acetyl-A-methylcholine aregiven in Table I. The ratio for acetylcholineilacetyl-ilmethylchotLne hydrolysisdetermined radiometrically on 18 tissue samples (6 brain tissues from each of 3 dogs) was5.21 ± 0.23; P= 0.95. The ratio so obtained with 1.0 mM substrate concentrationsagrees closely with one of 5.18 reported by Jackson and Apiison l for 0.75 mMconcentrations of the same substrates.

(C, Inldhbcion and radiaphospihaona bindbig folow7fig adtioi of •[3 2P]&'r-an to bmia heniogeatare- [Pj Soman in water was added in tile proportionof 1:99 (&,V/v) to the diluted homogerates of each brain tissue so as ti give finalconcentrations of 0.25 0-i m. o.so l0-9 M. 1. 0- I T9 M and 2.0" 10-9 m. The •mixtures were incubated for 2-3 hi at 37 so as to exceed 20 halllives for "'agn"(and presumably of dealkylation). Controls for ea:h tissue incubNi :n with water alonewere performed concurrently. Aliquots (0.1 ml I were removed fu assay of aceivlcholin.esteras, activitV usin. I- C|acel-tylmethylcholine 3s d.scribed above. Trichluroaceticacid was added to the remainder to 59- concontration. After 20 "t in trch. -oace tic acidsolution. 20 mi ol bo-ine albumin in 0.2 ml was added as a carrier. Conzrols c,..•tainingalbumin alone in the abscnce of tissue were also run. All samples were c ntrifugedat 2000 rv.!min and supernatants discarded. The residues cwre washed twice with5% tricifloroacetik acid. o'mce with ethanol- ether (3•:7. v/v). and twice with ether.followed by cenmrifugation and removal of the supematant after each wash. The protein-bound. soman-derved phosphorus was released by alkaline hydrolysis. and the 32F-labeled pinacolyl methylphosphonate and methylphosphonate. respectively, wer2 estimate:iby partition bet••en isobutanol-bentene (i:i. v/v) and an aqueous medium as previously

Biochinu Sophrx Arta. 235 i1971) 542-547

BBA REPORT

reported". The radioactivity contributed by methyl[ 3 2 P phosphonate was obtainedfrom M = Z - FAZ where M is the number of methyl 131 | phosphonate counts boundto protein, Z is the total counts and FAZ is the number of counts contributed bypinacolyl methyl[ 32 PI phosphonate. Substitutior; for the value of FA* in M = Zi(I - FA), gives At~ = Zj1 - 1.03 (R - 0.053)1(R + 1)j where R is the ratio of radio-activity in the organic solvent phase to that iti the aqueous phase. The counts wertcorrected for background and converted to tig by reference !o the radioactivity ofknown quantities of pinacolyl methyl 3 Pphosphonate and methylI32 PI phosphonaterun concurrently as standards. From the known welgis: of the tissue, and use of theabove formula, the amount of soman-derived pinacolyl methyl[ 32pi phosphonate andmethyli"PI phosphonate bound to protein could be calculated. At leveli of inhibitionbelow 50%, the contribution of pinacolyl methyl[ 32 Pi ph6sphonate to the total radi,-activty was usually less than I (k.-Since sufficient time for complete dealkylation ofsoman-phosphonylated acetylcholinestefase had been taken, the pinacolyl methylphos-phonate found was probably bound to sites other than acetylcholinesterase. andtherefore was not used for calculation of the enzymatic sites. Instead, the radiophos-phorus estimated as methy!j'IPl phospihonate for partial inhibition was extrapolatedto the values that would be expected for 100% inhibition (Table I).

A regression equation bet,%cn the valuts for control enzymatic activity toward•-[ I- 4 C. acetyl.-methyloline and methyl[,PI phosphonate bcund(Table 1) wasdeveloped. By use of thisequation, theoretical values for bound methyl1pP] phosphonatewre compared with the observed measurements. The correlation coefficient betweun thecalculated and observed 2lues for bound methylphosphonate was 0.99 with 95%confidence limits. This hch correlation suggests that radioactivity measured as methyl-phosphonate under these ,onditins is specifically related to acetvlcholinesterase activity.The turnover numbe.rs obtained for acetyicholinesterase from the ratio of 11

4C- acety!-choline hydrolysis to catalytic sites measured directly as soman-derived methyvi 3 P.-phosphonate (Table I) ranged from 2.4- I ý for zerebral cortex to 4.06- I O forcaudate nucleus. comparable to those obtained by lengthier, more indirectproceduresS""', and approaching values yielded by purified atce ylcholinesterase from-lectric ecil'1.

These findings further support the spelificlty of the measuremnint of catalyticsites in acetykholinestcrase with labeled soman under the conditions used ir this report.

In conducting the research described in this report, the investigators adhered to th-"Guide for Laboratory Animal Facilities and Care" as promulgat:d by the Committee onthe Guide for Laboratory Animal Resuw:es, National Academy of Sciences - Nati•n•aResearch Council.

R1EFIDREN.ZES

1 1.O. Midat and S. itrop.$. B&oL chem.. 190 (1951)119.2 W.?. AMtidg and A.N. Dwavion. BWUno t J.. 55 (1953) 763.3 SJ. Jandon, ILO. M![ic- N.. Schaffe, R. PEn and %H. summer•a. Dwcunim

FinNey SoC., 20(1955) 134.

*1k. dedvatiwa Of the fotmula f"r FA h giv in ref" S.

fUockfoL Xophy& Ark. 2 3S(1 -7 1)54 2-547

BOlA REPORT 547

4 8.3. Jandorf and H.P. NieNarnara. J. Pharnu.& EvptL TherAp. 98 (1950) 77.5K.NI. Wilson. Parwon Technical Paper. 398 !11954).Unclassdied.

6 R.L. Polak.5Aceta Phtydo izalDrmacot. .Veer. 1201963) 8..I EtA. Oosterlnan. LG. P.J. Warringt. H.S. Jansz, F. berends and J.A. Cohen. floe. 4t/a Intfern.

Comi'. Riochcnt. Vienna. 19.58. Perpamon Pre&, New York. i960. p.38.b 3.H.11. flsher and L.W. Hariris. lisoctiemn PharmacoL, 14 (1965) 64 1.9 L.W. Hlarris. 3.11. F'lesher. J. Clark and W.I. Cliff, Science. 1544 (19 66k 404.

10 3A.1. Fasei. L.W. Harris and P.T. Eserkouirz. Rsochren Pgarmtacoi.. !9 t1970) 42 1.li L.A. Loomis and B. SalAsky. Toxico!. App!.PamcL (936512 13.1. Couk% V.). Marsh and U. Read. IRhcMem . ýA. (1966) 861.12 fl.0. Miehel. &LC IHickley. LAI. Berkounr -and E U). Ilackley. Arch. Biochein. Btophy3.,

121 (1967)' 29.14 M.C. Ord and R.11.S. Thomrpwrn. Btochern. J'.. 46 '1950) 346.15 3.11. Fleisher. L.W. 1itrns and EU-. 2uri'. .j. Phartnaco!. tsp:! ac.15(96)3.16 AXN Sukotuos. M.G. Friher't and R. Ilesra. Egocnein Med. 3 G(9196¶8 1.V! R.L. iackson and %,.H, Apnison. J. &'urn-efln, 13 (1966) 13-51.18 JA. Cohien and t.O.P.J. War~inpz, fliorhar. Riophys. Acm. 11(1953) 52.19 Il.C. Lasicr.J. Rio!. Chrem. 2316% t96i) 2296.-

fliac/am. Bfioph vs Am,. 235 (1971) 542-1547

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