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Assessment of efficacy of octivated charcoal

for treatment of acute T-2 toxin poisoning flh11 FILE ropy

Robert F. Fricke and Juan Hl. Jorge

PRFORMIG ORGANIZATION 1404 AMC 0,000911 PQ ItA Lgwer.t 000itci. tA"i^1A A WORKN WsT? 4~WS06

US Armay Miedical Research Institut3 of InfectiousDiseases., SGRD-UIS-D

FotDtik rdrc.MD 21701-5011 ~OYO?

US Army Medical Research and Z'~velopment Command 14 November 15MiIS. "U"SGIEf, OP Rs)

14. i*OXSIT01GMQ AGENCY MAMIE A A0ONZS.S( 1f Etsr *0 CAMORdlim4 OM.0) IL S*Cu~jTY "ALA (of 00* mae

~-s ~ 14L DISTftIUUTION STATEMENT (of &e Amato

iliu A"Distribution unlimited -approved for public releaser

14L SUPPLEMENTNARY NOTES

To be published in Journal of Toxicology -Clinical Toxicology

19. KEY WORDS (Centbm. do toeme .Wd* Ifft**F 40d 140Rf OF~tt 61' U AN

M 2 AWTVIAC? (=wC wýmvmsd i esm ON 10 1g ti &V Wleek 004m)

T-2 toxin Is a fungal metabolite which can cause death or illness upon inges-tlz- As a potential antidote, activated charcoal was assessed for efficacyin decreasing the lethality of both oral and parenteral exposure to T-2 toxin.In vitro binding studies, using the Langmuir adsorption isotherm, showed thatactivated charcoal had a maximal binding capacity of 0.48 KS toxin/mg charcoaland a dissociation constant of 0.078 ag charcoal/i. In vivo, orally adminis-tired, activated charcoal was assessed for treatment of ac-ute oral or paren-

Lteral-exnosure to T-2 toxin in mltp- Aft-or nra1 t-nlrn a ln-epp4n,, f~zjk&1IDD F~cwm,, IM3 =man aw I~ Nwov as aOEOLETS

SECUOirY CL.ASSIFCATION OF ThIS PACE (Me 0010 ENCOM4

6RCURMTY• LASSIPICATION OF THIS PAO•geW' I DWP RW6I,

untreated mice shoved only 62 survival after 72 hr. Charcoal treatment(7 g/kg, po) either immediately or 1 hr after toxin exposure resulted in signi-ficant improvement in survival, with values of 100% and 75%, respectively.After parenteral toxin exposure (2.8 mg/kg, sc), untreated and chArcoal-treated(7 g/kg, po) mice showed 50% and 90% survival, respectively, after 72 hr. LD50value for T-2 toxin, eetermined at 96 hr after intoxication, increased signifi-cantly from 2 mg/kg for untreated controls to 4.5 mg/kg for act ited charcoaltreatment.

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ft. Detrick, Yrre~rtek, Plarylaiad 217al-501I

T-2 toxin is a fungal astabolItt which tse ta•** death orill.068 Upoa ifteStiOn. AS a pOt*Atial Sotidote, actiaVt#4 Car-coal vas assessed for efficacy ia decroaeiag the lethality of botboral and partnteral e*posure to t-2 toiae* ta vitro biodt"studies, v.aing the Leogmair adsorption liotherm, so%4d that oetiowated charcoal& had a m.xisal binding capacity of 0.4$ ) toiniogcharcoal and a diseociatioe constant of 0.078 m chsrcoel/l. I*v'v..., orally adainisterod, activated chbarcoal waa aatoe#e4 tottreastmant of acute oral or parsateril eaVoe•re to T-2 tosts ismice. After oral toala adlaistratiom (5 mg/k!), %ntreated idesshowed only 6% survival after 72 hr. Charcoal treatmat 07 #/k4,po) either immediately or I hr after toui *xposvr- respite4 !%significant imrovemt it survival, with vluoes of IOCL and 7'•1,resptctively. After parenwteral toxin expoesur (2.8 ag/k.;,sc),untreated and charcoal-treat*ed (7 S/kg, po) mIce showed 5oIt &a90% survival, respectively, after 72 hr. tP.0 vale* for T-2toxin, determined at 96 hr after intoxication, increased igtti-ficantly from 2 m/k4 for untreated controls to 4.5 q/klg foractivated charcoal treatment.

88 1 19 014

INTRODUCTION

ST-2 toxin is a toxic secondary metabolite produced by a

variety of Fusarium fungi-i. This trichothecene mycotoxin is

highly toxic and has produced illness and death in humans and

livestock after ingestion of food or grain contaminated with fun-

gal toxins. Oral exposure to T-2 toxin has profound effects on

rapidly dividing tissues j1 Thus, the spleen, testes, thymus,

bone marrow, gastrointestinal tract, and hair follicles all show

significant necrosis "3•* .2

One of the most consistent and well-defined effects of T-2

toxin in several animal species is the dramatic cytotoxic changes

in the gastrointestinal tract ,--41. After parenteral 13r-topi-

cal IU 06r oral flexposure, there is severe and prolonged diar-

rhea I*T' accompanied by increased intestinal permeability 'tTf.The severe gastrointestinal effects of T-2 toxin may be a

result of the preferential excretion of T-2 toxin metabolites via

the bile into the intestine . Activated charco.il might be ef-

fective in decreasing lethality by binding toxin metabolites in

the gastrointestinal tract. The toxic compounds would therefore

remain in the gastrointestinal tract, decreasing the reabsorption

via enterohepatic recirculation. In the studies presented here,

efficacy of activated charcoal for treatment of both oral and

parenteral toxin exposure was assessed. '--3 +

MATERIALS AND METHODS

In Vitro Experiments

In vitro adsorption experil its were performed to determine

the equilibrium binding and diss, ;Aation constants for T-2 toxin

and activated charcoal (Amoco AX-21, Anderson Development Co.,

Adrian, Mfich.). The charcoal used in these experiment3 was highly

activated and has been shown to be superior to other forms of ac-

tivated charcoal for treatment of a iariety of intoxications [9].

A stockh o4A~ f T-2 tot1a. )1* Purttw

chwot~rtfi.1, 00.)*.. v ~~ roesr#4 *Imt at4 a *t ~#f

of to Ktr 1 1*#*t* ;)

After 1-(iVutt~q at ro,. tomprt-si*., frov s4 b4 t~alv,

0 ;"to4 ) L.L tadoa ytttl w*O 4e'#tovv ý is am 611,*t tt tse Wc*rt

Wsant firo*ttolm.

*Wusat of tre., C (04 tcatie/t, **d *0404, Q (go t~tm/og

ctarco~l). torts wars, calctolat94. No 4*t. wero *al"yee4 Vy the

Vo vaa o o*i f K. the *q'.tltbctvm lotf4t*. e*ots*t, wr~ the

SAe1tIMM btniIMS C&aadtt (Q# teitt/*$ ChetC.1), Vote 4EctV*4

aft.r ItfWar rogreselo. saslysts of the "kvvo "Uttea%.

PAl. Sice ($Wife ICS). VIajM'tft 21-14 *. *4*rc misjt.1Ad aI

controll*4 tompratiare in-2C wi.re-'Sottom cop* &m4 sIllw.$~f*,d RAJ "etot ad 11It Se.The.,k toetit *qiatt,,,q vo 01,de4

with propyl.tne glycoli-.thia,46 ($1:10) s0d 100 *1 Was mo4a1otoetr*4

either o&tbcuatsaoouly or orally to t6-hr tasted sic*. 4ct i~ot.1

charcoal was *uvp~fd*4 to diettil1* woter *4d odstistotrv-d by!

Saag tit a total role it 5WX al*

Maggio=

The number of surviving mice was determined at different

times after administration of T-2 toxin. Data were analyzed for

statistical significance uping the Fisher's exact test [111. LD5 0

values for T-2 toxin were calculated by the probit regression

analysis based on best fit common slope [121. Statistical signi-

ficance of the LD5 O values was determined by least significant

difference analysis on the pooled variance of the LD 5 0 values

[13).

RESULTS

In Vitro '~xperiments

Langmuir isotherm (Figure 1) was used to calculate the maxi-

mum binding capacity, Qm, and the dissociation const'ant, K, for

the adsorption of T-2 toxin onto activated charcoal. The calcu-

lated values (* S.D.) for Q. and K were 0.48 * 0.136 mg toxin/mg

charcoal and 0.078 * 0.0197 mg charcoal/i, respectively.

In Vivo Experiments

Efficacy of orally administertd charcoal for treatment of

oral toxin exposure was asiessed in mice. Mice were either un-

treated or treated with activated charcoal at the same time as or

1 hr after challenge with T-2 toxin (5 mg/kg), po). The number of

surviving mice was determined over a 72-hr time course (Figure 2).

The percent survival of mice in the untreated group declined

steadily throughout the observation period, reaching a value of 6%

ý'-"i•ter 772"hr. All of the mice in the group treated immediately

with activated charcoal were prctected against the lethal effects

of T-2 toxin. If treatment was delayed for I hr, the percent sur-

vival was lower than that of the group treated immediately. The

percent survival of animals in the delayed treatment was, however,

significantly higher than in the control group.

•MM-IIr.XUA-

&aW.0totor#4 T-2 toale Wee also aaSovood. t1"Wt.1#I foll~Oui a

paqslmltctliu of T-2 titale (2.6 *44jý atr. were ot,,týr

left antreatod or treated witti art ivated chaco~l (U II&g). The

mieSw~r 24 aurviIng~ sic* was agaili *rte~rsl ',y~r 72-tr (71#vr*

3). The porcefit of #urvvltmn. wtce to e vOw treet.44 gromip *so 93,.

Wbc Was *O sLSGOLCA 7 :040 thee th0 T4106s Of 90 fot the

trsett* g5#%p.

becoate oral chaercoal Malmtnstratios ma, effective, La

decogongthis lethality of *vbcutanmoo*4 tovis #tpomva~r, IZ

va1ltes foer T-2 toxin war* dvorl~od ftý- jntroat*4 and cbarcoai-

trvat*d sice. T~he L.ý5 Values were, cal#.ulat*d at Vi4-r iotategals,

after toals injection far a total of 96 ht (fipt# 4). At all

tius p~kintts. thS LDý, VaiWee tot the a%41CO*-trSat#4 &iLCO were

significantly higher thal for the umtr**ted c04trols. W5 uaos,

for ustratted aito dtcreased froe a 24-4ir LD5o value of aboat 3

agfkg to a voliae of I mgbg at % hr. The chatcoail-treated sice,

con the other han, 'Aid ftt shw a 91isir del1oo to LOSO Vato*#*

Thromghost the otoeervattoe pieriod, the U50velues toT charcoal-

troat'd aico weaeim*d C~netsaut at apritetey 05 .6/k;.

Activated charcoal has beem used for the trestimat of a wide,

varitty of Istollcatione. Aflatoicoes is to $at$ j14J and

chIckens (151 has been treated eucc~essfalLy with activated thar-

,coal. for treatment of T-2 tosicosie, there are several report#

!* the, literature which ouflet that a~dsorbint pagete is the gam-

tetontiestinal tract sight be *ffe-tive astidotee. The pi~sestaof at-

intestinal roughalt offectively altered both the toxicity a&M os-

crttloa :)f mycotoxins. 4ittA~al Ltitovicatod with tither 1-2 toxin

or zoaralenone and 4ThleiIj%#tiy fed ditto high im cr~isd alfalfa

fiber f16,171 or bento-itto (III not only shoved increased

SO

survival, but had improved weight gain and feed consumption as

well. As the percent of fiber in the diet was increased, there

was a proportional increase in the fecal, but not urinary, ex-

cretion of toxin (19,20]. "These data suggest that other, more

effective adsorbing agent&, such as activated charcoal, might be

effective as well.

Activated charcoal was evaluated for efficacy on treatment of

acute T-2 toxicosis in mice. Based upon in vitro experiments, T-2

toxin was found to be tightly adsorbed onto activated charcoal,

vith biiding and dissociation constants selilar to those reported

for nefopaa [101. Since activated charcoal was effective in

vitro, further in vivo experiments were carried out to assess its

efficacy in treatment of oral and parenteral toxin exposure. For

oral administration of charcoal and toxin, if treatment was

initiated immediately after toxin exposure, there was a 100%

survival of the mice. If treatment was delayed up to 1 hr, there

was still significant, but lower, survival.

Perhaps the more important finding is the protective effect

of activated charcoal against parenterally administered toxin.

Following subcutaneous toxin administration, oral administration

of activated charcoal resultcd in significant improvement in the

proportion of surviving animals and a significantly higher LDS 0

value for T-2 toxin. Parenteral exposure to T-2 toxin results in

both free and conjugated metabolites appearing in the bile (81,

which may be reabsorbed via the enterohepatic recirculation. The

presence of adsorbing agents in the gastrointestinal tract

effectively binds the metabolites, thereby short-circuiting the

enterohepatic cycle.

In summary, we found activated charcoal to be an effective

antidote for treatment of T-2 toxicosis in mice. Survival after

oral or subcutaneous toxin exposure was significantly improved by

charcoal treatment.

ACKNOWLEDGMENTS

Thanks are due to Dr. J. G. Pace and Dr. K. A. Mereish for

their advice during the writing of this paper, and to Patricia

Weicht for preparing the manuscript.

FOOTNOTES

1. In conducting the research described in this report, the

investigators adhered to the "Guide for the Care and Use of

Laboratory Animals," as promulgated by the Coamittee on the Care

and Use of Laboratory Animals of the Institute of Laboratory

Animal Resources, National Research Council. The facilities are

fully accredited by the Azerican Association for Accreditation of

Laboratory Animal Care.

The views of the authors do not purport to reflect the

positions of the Deparcment of the Army or the Department of

Defense.

2. To whom correspondence and reprint requests should be sent.

REFERENCES

1. J. R. Bamburg and F. M. Strong, "12,13-Epcxytrichothecenes,"in Microbial Toxins VIt (S. Kades, A. Ciegler, and S. J. AJl,eds.), Academic Press, New York, 1971, p. 207-292.

2. S. C. Lee, J. T. Berry, and F. S. Chu, Immunoperoxidaselocalization of T-2 toxin, Toxicol. Appl. Pharmacol., 72,228-235 (1984).

3. J. Palti, "Toxigenic Fusaria, Their Distribution andSignificance as Causes of Disease in Animal and Man," in ActaPhytomedica, (J. Colhoun, H. Kern, and H. Richter, eds.),Verlag Paul Parly, BerLin, 1978, p. 58-93.

7

4. M. Saito and T. Tatsuna, "Toxins of Fusarium nivale," inMicrobial Toxin, Algal and Fungal Toxins, VII, (S. Kadis andA. Ciegler, eds), Academic Press, New York, N.Y., 1971, p.293-316.

5. H. B. Schiefer and D. S. Hancock, Systemic effects of topicalapplication of T-2 toxin in mice, Toxicol. Appl.Pharmacol.7.6, 464-472 (1984).

6. Y. Matsuoka and K. Kubota, Studies on mechanisms of diarrheainduced by fusarenon-X, a trichothecene mycotoxin fromFusarium species, Toxicol. %ppl.Pharmacol. 57, 293-301(1981).

7. Y. Ueno, "Trichothecenes: overview," in Mycotoxins in HumanHealth (J. V. Rodericks, C. W. Hesseltine, and M. A. Hehlman,eds.), Pathotox, Park Forest South, Ill., 1977, p. 189-207.

8. J. G. Pace, M. R. Watts, E. P. Burrows, R. E. Dinterman, C.Matson, E. C. Haeer, and R. W. Wannemacher, Jr., Fate anddiotribution of H-labeled T-2 mycotoxin in guinea pigs.Toxicol. Appl. Pharmacol. 80, 377-385 (1985).

9. W. B. Buck and P. M. Bratich, Importance of activatedcharcoal in veterinary practice, Vet. Ned. submitted forpublication (1985).

10. P. J. Neuvonen, M. Kannisto, and S. Lankinen, Capacity of twoforms of activated charcoal to adsorb nefopam in vitro and toreduce its toxicity in vivo. J. Toxicol. Clin. Toxicol. 21,333-342 (1983-84).

11. S. C. Gad and C. S. Weil, "Statistics for Toxicologists", inPrinciples and Methods of Toxicology" (A. W. Hayes, ed.),Raven Press, New York, NY, 1982, p. 291-292.

12. D. !. Finney, Probit Analysis. Cambridge University Press,New York, New York, 1971.

13. R. G. D. Steel and J. H. Tcrrie, Pr•Lciples and Procedures ofStatistics, McGraw-Hill, New York, 1960.

14. R. C. Hatch, J. D. Clark, A. V. Jain, and R. Weiss, Inducedacute aflatox~cosis in goats: treatment with activatedcharcoal or dual combinations of oxytetracycline, stanozolol,and activated charcoal, Am. J. Vet. Res. 43, 644-648 (1982).

15. R. R. Dalvi, and A. A. Ademoyero, Toxic effects of aflatoxinB in chickmns given feed contaminated with Aspergillusflavus and reduction of the toKIcity by activated cnarcoaland some chemical agents, Avian 9is. 28., 61-69 (1984).

16. M. S. Carson and T. K. Smith, Ltfect cf feeding alfalfa andrefined plant fibers on the toxicity and metabolism of T-2toxin in rats, J. Nutr. 113, 304-303 (1983).

17. T. K. Smith, Influence of dietary fiber, protein, and zeoliteon zearaierone toxicosis in rats and swine, J. Anim. Sci. 50,278-285 (1980).

18. M. S. Carson and T. K. Smith, Role of bentonite in preventionof T-2 toxicosis in rats, J. Animal Sci. 57 1498-1506(19e3).

19. T. K. Smith, Effect of dietary protein, alfalfa, and zecliteon excretory patterns of 5',5',7',7'-H zearalenone in rats,Can. J. Physiol. Fharmacol. 58, 1251-1255 (1980).

20. L. J. Jame and T. K. Smith, Effect of dietary alfalfa onzearalenone toxicity and mtaboli~m in rats and swine, J.Anim. Sci. 55. 110-118 (1982).

FIGURE 1. Langmuir isotherm for adsorption of T-2 toxin ontoactivated charcoal.

FIGURE 2. Effect of activated charcoal on the percent survival ofmice after oral T-2 toxin exposure. Mice were all gavaged withT-2 toxin (5 mg/kg) and either untreated (closed circles), treatedimmediately (open circles), or 1 hr later (squares) with activatedcharcoal (7 g/kg). Significant differences from untreated controlvalues are indicated by * (p < 0.05), ** (p <0.01), and *** (p <0.001).

FIGURE 3. Effect of activated charcoal on the percent survival ofmice after subcutaneous T-2 toxin exposure. Mice were injectedwith T-2 toxin (2.8 mg/kg) ana either untreated (closed circlea)or treated with activated charcoal (7 g/kg) (open cirlces).Significant differences from untreated control values areindicated by * (p < 0.05).

FIGURE 4. Effect of activated charcoal on the 05o value forsubcutaneously administered T-2 toxin. LDSO values weredetermined for untreated (open bars) or activated charcoal-treated(croashatched bars) mice at times indicated. Significantdifferences from untreated control values are indicated by * (p <0.05), ** (p <0.01), and ** (p < 0.001).

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