7AD-M193 744 THE METADOLISM OF NITROG URNIINE AND NITROSOOANIDINE 1/1BY RAT HEPATIC SUDC.. ( U) LETTERMAN ARMY INST OFRESEARCH PRESIDIO OF SAN FRANCISCO CALaCASIFIED0 P I SIMBOLI ET AL. JUL 97 LAIR-S7-?0 F/0 61 M

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Laboratory Note No. 8 7-70 6U 11987

03 The Metabolism ofNitroguanidine and Nitrosoguanidine

byRat Hepatic Subcellular Fractions

Paul B. Simboli, BS, SGTBert Ho, PhD

andDon W. Korte, Jr., PhD, MAJ MSC

TOXICOLOGY BRANCHDIVISION OF COMPARATIVE MEDICINE AND TOXICOLOGY •

D[STRIBUTION SZTEMLNr X]Approved tot public relecuelg

Dtjazibution Unlimited

JULY 1987 Toxicology Series 152LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

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The metabolism of nitroguanidine and nitrosoguanidine by rat hepaticsubcellular fractions--Simboli et al.

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In conducting the research described in this report, the investigation adhered to the "Guide for theCare and Use of Laboratory Animals," as promulgated by the Committee on Revision of the Guidefor Laboratory Animal Facilities and Care, Institute of Laboratory Animal Resources, NationalResearch Council.

This material has been reviewed by Letterman Army Instituteof Research and there is no objection to its presentation and/or publication. The opinions or assertions contained hereinare the private views of the author(s) and are not to be con-strued as official or as reflecting the views of the Departmentof the Army or the Department of Defense. (AR 360-5)

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Laboratory Note No. 87- 70 ss 4

4. TITLE (,ind Subtil) 5. 7YPE OF REPORT & PERIOD COVERED

'11e fiet, lhba i.,if of I ait ropuaniline and nit roso-giidino by JiewI-:It hiepat ic suhee i hda r I ric ti' on

6 ERFORMINO ORG. REPORT NUMBER

7 AUTtHO' ICONTRACT OR GRANT NUMbLR(.)

P1ffl I . -Si 1ba6,'~ ,Gl BerIt 110, PAm a ~.urrtc , ;I"P1hIl, MXIj NISC

9. PERFONIMING C)RGA',I C ATiON NAME AND ADDRESS ICPRO RAM ELEMENT. PROJECT, TASK(

!ivisicil of ie'J_iologyLeOttermlan uuy\ lns titute of Research

I Presjii of S:i Francisco, Q\ 94129-6800III. CONITF ,LIIG OFFICE NAME AND ADDRESS 12. REPORT DATE

July 198713. NUMBER OF PAGES

14. 15ITNN GNYNAEAAOESI ifeetIoCiIoIrgOtc)I. SECURITY CLASS. (of this report)

Unclass if LedlIS, OEC..ASSIFICATION/DOWNGRADING

SCR FOULE

6. DIbTI 6IAU'ION T A f, ti, N T (,,I this .Nep.riI

\ImdIi sa reeae isr i hut i on ji ul i IIi I el .

7. DISTRiBUTIOlI STAT I M _: 7 tfhe jbstrac.; enered In W~ork 20, If Irrn I,,,R'o l

_T8 SUII ~ ,.TA-e

20. 'O TRAC-T 'Conrff': ,, ' r, ,,I- it nec..y id id-,n' by block ri-nb.r)

lie~'tib.i it roguan id ie CGI and ni rs)AI e(W crat

he~~I! fr .. ,. ic tions was evamiiic . The aiC1501fiet ioll and 10,000;;i1tAI'1!I uInt rea;ted an1d plictnolmrbi tall - t reat 12, animlals and the 9,000

X 2 '1't an' n ".\ Nrochi or -induced rats were used( Jn Cr t h is st udy . The invitro ie t ahol i .i2 4! -,lh5t rate a eaue MIS h\' 1,. '11e resu;Llts; ili di eat e t. lt

1w' it K' i NG nor Are met aboa Ii .ed by thte hepbot ic tll hlma fI r'at ions,,.

DD cA." 1473 11 1TT 1f .0V AS I S OBDSO L FT E ;

Phe mretabolisin of nitroguanidine (WJ) andnitrosoquc-,.iiine (NsG) by rat 'hepatic subcellular fractionswas examinedI. The microsomal fraction anI 10,000 x g

supernat-ant 'From untreated and3 phenobarbital-treated I

animrals arC' the 9,000 x g supernatanL from Arochior-inclucelrats -were usedl for this study. Thro in vitro metabolism ofsubst-ite vi~s measured by HPLC. The results inaicate thatneithor NG nor NsG are metabolized by the hepaticsubcelluilar fractions.

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Simboli et al--i

The Metabolism of Nitroquanidine and Nitrosoguanidine byRat Hepatic Subcellular Fractions--Simboli ot al

Nitroguanidine, a primary component of US Army triple-base propellants, is now produced in a Government-ownedcontractor-operated ammunition plant. The US ArmyBiomedical Research and Development Laboratory (USABRDL),as part of its mission to evaluate the environmental andhealth hazards of military-unique pollutants generated byUS Army munitions manufacturing facilities, conducted areview of the nitroguanidine data base and identifiedsignificant gaps in the toxicity data. The ToxicologyBranch, LAIR, was tasked by USARRDL to develop a geneticand mammalian toxicity profile for nitroguanidine, relatedintermediates/by-products of its manufacture, and itsenvironmental degradation products. Nitrosoguanidine is apotential envirn)rlmental degradation product ofnitroguanid ine.

The development of a toxicity profile for NG and NsGwill aid in understanding the safety or hazards of thesecompounds. Metabolic fate studies are an integral part ofthese toxicological tests. To supplement in vivo studieson the fate of nitroguanidine in the rat, this studyexamines the in vitro metabolism of NG and NsG by hepaticsubcellular fractions. Additional impetus to examine thein vitro metabolism of NsG is provided by preliminaryreports that NsC was mutagenic in mouse lymphoma assayswhen activated with Arochlor-induced 9,000 x g supernatant(9K) (1).

The objective of this study is to quantitate the extentof metabolism of NG and NsG by three rat hepaticsubcellular fractions: untreated and phenobarbital-treated10,00 x g supernatant (10K), Arochlor-induced 9,(0 x gsupernatant, and antreated and phenobarbital-treatedmicrosomal pellet. If the substrates are found to bedegraded metabolically, the metabolites will be isolated,identified, and quantified.

MATERIALS AND METhODS

Chemicals

Glucose-6-phosphate, g]ucose-6-phosphate dehydroqenase,NADP, and Fischr's medium for leukemic cells of mice were

-- K . .. K.., " W 4 " ,'

Simboli et al--2,

purchased from the Sigmia C'lemical Cro., t-1u; ~Nit trogu an irine was supp I ioe' by the Suin F I'~ Xim~it io IIPlant, Desoto, KS. The UIS Army Biome,!i cal I -in 1Development Liboratory (USABPDL, Fort Petric j nro)vi-iedthe NsG . The Arochl~or-i ndiicedi I1 s upe rrr ' , pi'by Microbiological Associates, Retli-s; 1l, -I.

Instrumentation

The E3PLC system consistedl oF two T-V" .0~os:Olleto an LKB 2]r)2 HPLC controller; 20-ul irw-c i::s wc-re m~Olvia a Waters Wisp 710AP., the lot -etor wv1- I1Spectroflow "/73; the inteqrator v.i '~ '--P?k. Th eHPLC column w ,as a Brownlee C-1iS Sphceri col le ( 4K mm r~ x2 5 mm) , a mob)il1e pha se o f 10 1 M -eul !? S ti 1 72 ati Ir lOwrate of 0. 7 ml] /min; the, detec-t!or se*, t -i t1 n-,) nAInFS) . The c-rnt rifuqec; il wori' i SC'r 1, 1Sorva I I RC-- 1; ur-erspeei Po f r i-' roit (,Ic'l ;P v', rv1

Heckman TLr-7' 1 trcrir p A nrm 'I

homoge ni~ze r L.' u' r1 te oiq (zet 1 '

Animals

Male albino Sprariae-Dza'Jley ratF, w%1-.2r eobtained frort P-:-ntir.-Kinqm 0i, Fremiont, "n. ' ;mcnagenit sodium plhonobarbi tol (P)wais 10,; n -Io i n(8mg/kg) -or f our conre-cut iv(, 'iays pr ior to- sar i f i e on d~ay5. The untreaited animals were Iof f unt reo t -,ri rl 'Iiistime period. Animals were fasted for a 24 houo perinodprior to sacrifice (SOP-OP-STX-88, 'Preparation of HepaticSubcellular Fractions"). Th-e rats were sacrificed bydecapitation and the livers were rapidlIy r ,iro-l anol ri oseclin cold 1.15% KC1. The livers were bl-otted iry, wihland placed in a volume of cold O.lMI Tris !ioffec k!-pH 7.4)equal to twice the combinedl weight of thie livers. 7nelivers were minced with scissors and homeo-jon i o-.7, 'Tehomogenate wis centri fugecl at 1(,10 ~h~lx ( (1-1' f-or -1mrintorn The- 10~,000 x (I snpernatant -'? ' 'o C frqr'.iat 100,000~ x qj for r(f' mrinutes(0-iC) Tn

u Itra c;nt r t i( jaQt-i'on, the x'~ i~ p- nor .11i Sliscarded -~,j t~~r' ~ nell ',t~ sr ~ 'v'."i

hilf f'r 52.~ ml 1,f of Ii-? r) . 'he t)t1'j

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Simboli et al--3

untreated IK supernatant, PB-induced microsomes, untreatedmicrosomes, and Arochlor-induced QK supernatant. The 1OKsupernatant and microsomal incubations were conducted at aprotein concentration of 4 mg/ml. The 2-ml incubatescontained 1.0 mM NG or NsG (2 mg/ml DMSO), 5 mM glucose-6-phosphate, 5 mM MgC.; I unit/ml glucose-6-piosphatedehydrogenase, 0.IM 11 7.4 Tris buffer, and the liverfractions. Incubates also contained (.5 mM NADP whereasthe control incubates did not. Additional experiments inwhich the enzyme system was heat-inactivated were conductedby immersing the subcellular fraction in a 900 C water bathfor I-15 minutes before adding the other cofactors.

In order to simulate the activating con(ditions formouse lymphoma studies, the 9K incubations contained, in atotal volume of 2 ml: 8 mg of protein, 1.0 mM NsG, 3.375mg/ml isocitrate, 9.6 mg/ml NADP, and Fischer's medium forleukemic mouse cells. Control incubations did not containNADP. Control incubates containing only Fischer's mediumand NsG were also prepared.

All incubations were performed in a water bath shakerat 370C. The Incubation period for the 10K and microsomalfractions was 20 minutes. The qK supernatant fractionswere incubated for 2 hours. After the desired incubationperiod, the postincubates were placed in an ice bath andtPrminated by the addition of 250 ul of 5% ZnSO ; 2 umol ofthe internal standard (IS) methylnitroquanidine 4 was thenadded. The incubates were centrifuged for 5 minutes at4,000 g, and the resulting supernatant was filtered viaMillipore Swinne: filtering system (0.45 u). The filtratewas analyzed via IHPLC.

RESULTS

The HPLC traces obtained from the postincubates wereclean with no interfering peaks. Nitroguanidine eluted .sharply at 4.9 min, NsG eluted at 4.5 min, and the internalstandard eluted at 5.8 min. Table 1 shows the HPLC peakarea ratios of NG/IS obtained from incubations ofnitroguanidine with untreated and PB-induced 10Ksupernatant and untreated and PR-induced microsomes. Nosignificant difference in the amount of NG remaining inincubations with the NADPIH-generating system and in thecorresponding control incubations without NADPH was seen.The same results were obtained when the control incubateswere heat-inactivated. The standard deviation values givea rough estimate of the precision of the analysis7 in thecase of NG, the precision is about 5%.

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Simboli et al--4

Table 2 shows the HPLC peak area ratios of NsG/ISobtained from incubations of NsG with untreated and PB-treated 10K supernatant and microsomes as well as withcommercially obtained Arochlor-induced 9K supernafant. ","

significant differences are seen in the amount :present in incubations with or without NADP a'- o • again,the same results were obtained when the enzy1ites cf thecontrol incubates were heat-i niactivated. Addi I io!','1 ly, Ws1-1y

was not degrade( over a longer incubat ion pc1, C ( ) hol!rs;at 370 , results not shown). In this cas-, Iheo-eisionin the measurement of NsG appears to be in the rder of 5-

DISCUSSION

The metabolism of NG and NsG by untreated andlphenobarbital-treated rat hepatic l0,00 x g superriatantand microsomal fractions was examined. The disappearanceof NG or NsG from these incubations was monitretd l 1y I3PLC;the results indicate that these compounds do not minereeNIDPH-dependent metabolism by rat liver 10,00 x isupernatant or microsomes. This observation i- ccnsist(enwith recent in vivo data that indicate NG to I,- ri-lyabsorbed through- th_ e gut, to enter the bloodstroa:n, ctfni tobe quickly excreted unchanged into the urine; it ;i.apearsthat NG and NsG are not metabolized by the ',er but irerapidly passed! out of the body (2). No me - , , i::o k;:i. ','nin incubations conduicted with hepatic fractions O . i (,, I

from PB-induced animals, thuf4 demonstrati ne ta,-i!- :n,l Nsare not subject to hepatic enzyme biotransforrq-t-V.

Nitrosoguanidine was found to have slight mutaegniccapabilities in mouse lymphoma assays (1). The metabolismof NsG was examined under conditions similar to that of themouse lymphoma assay with Fischer's medium for Loikomiccells of mice and OK supernatant to determine if vicetabol icactivity could assist in explaining NsG mutagenic-ity. NoNADPH-dependent metabolism of NsG was seen in this system.This seems to indicate that the inutagenicity oinervd inthe moii--e ]ympho:na assay is not due to a metabolic archemical ,Iegiradation product of NsG. However, it shoul.1 benot,-] that, dli,? to the imprecision (CV is ]1,-ts: than 7 ') ofthe assay in q.antitating NsG. small amount,- (tit- o 1t0 ug)

f ,sG may he mtabo] izod and yet co inoet?-ctl unilr these

1 ikll t tions a ri.onal Iy, possible metaho] ires of P:,a-eV kl t o 1 % - o" ,'1' m ),r 1 '-X t laIct ian co )eFffi,-i -n r . t1I Iy

1'u ' 1, ,i ftf-IC I 11 o o e t It I n V It . - ,<

Simboli ot al--5

REFERENCES

1. 'larbell JW. Personal communi,cition.

2. Ho B, Tillotson -, inc.nnon .- , Simboli PB, Korte D;.The fate of nitroganl'iine in the rat. Toxicol AppIPharmacol (in preparation).

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C.

TABLE I

The NADPH-Dependent Metabolism of Nitroguanidine byUntreated and Phenobarbital-Treated (PB) Rat Hepatic

Fractions

Peak Area Ratio of NG/IS + SD (n=7)

+NADP -NADP

10K Supernatant

Untreated 1.10 + 0.01 1.12 + 0.01PB 1.07 + 0.04 1.03 + 0.06

-C.

Microsomes

Untreated 1.02 + 0 .0 2a 1.03 + 0 .01 b

PB 0.99 + 0.02 0.99 + 0.02

a N= 6b N 5

.-.

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D.

Simboli et al--7

TBLE. 2

The jIDpH-Deperdent Metabolism of Nitrosog anidine by

Untreated, Phenobarbital-Treated (PB), and Arochloro-InducedRat Hepatic Fractions

'.4'

Peak Area Ratio of Nsq/IS + SP (n=7)

+NADP -NADP.

I.V Supernatant

Untreated 1.00 + 0.09 1.00 + 0.01PB 0.P6 + 0.06 0.86 + 0.07

Microscmes

Untreated 0.89 + 0.02 0.99 + 0.01PB 1.13 + 0.05 1.37 + 0.02

9K Supernatant 0.96 + 0.01 0.93 + 0.01- - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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