Table Of Content;
PATHOLOGICALCHANGES INMURINEHEARTS INDUCEDBY INTERMITTENT
ADMINISTRATION OFCIGUATOXIN
KIYOSH! *reRAO.EMIKOITO, MISAKOOHKUSU ANDTAKESHI YASUMOTO
Terao, K., Ito,E.,Ohkusu,M &Yasumoto,T. 19940801: Pathological changesinmurine
hcaru induced by intermittent administration ofciguatoxin. Memoirs ofthe Queensland
Memoirs34(3): 621-623.Brisbane.ISSN0079-8835
Ciguatuxin (CTX) atdoses of0,1 or0,05u,g/kg were given orally by intubation into male
ICR miceonceaweekfor25weeks(0.1fig/kggroup)or40weeks(0.05u,g/kggroup). Until
about 10 weeksafterthe beginning ofthe experiments the mice in both groups showedno
abnormal clinical signs. Afterabout 18 weeks, mice treated with0.1u,g/kg showedmarked
hypertrophyofthehearts;nopathologicalchangeswereseenintheheartsoftheothermice.
Therewasswellingorruptureoftheendotheliumofthecapillariesand wideningcausedby
exudation orcollagen fibers in the interstitial space. Occasionally, degenerated of swollen
mitochondria wereprominent in the myocardium. Accumulationsofplatelets in thecapil-
laries were frequently observed, Mice treated with low CTX dose showed no pathological
changeseven attheultrastructural leveluntil40weeks. tHUSCTXhasapotentcumulative
effectonthecardiactissue.
Kiyoski Tvrao, Emiko ho & Misako Ohkusu, Research Centerfor Pathogenic Fungi and
MicrobtalToxicoses,ChibaUniversity. I-S-l,Irtohuna, Chuo-ku.Chiba260,Japan;Takeshi
Yasumoto, FacultyojAgriculture, Tohoku University, 11 TsutsumidoriAmemiva,Aoba-ku.
980Sendai, Japan; 2 February 1994
Ciguatera is one of the most serious tropical 23g) were obtained from Charles River Japan
food poisonings of fish in the vicinity ofcoral Inc..Tokyo. 40mice weredivided into 3 groups,
reefs;itssymptomsareneurological,gastrointes- Group I: Five mice were given physiological
tinal and cardiac (Baden,1983). Several toxins saline with a stomach tube once a week for 40
were isolated from contaminated fishes or cul- weeks and servrxi as ContcoL Group 2: Twenty
tured dinoflagellates and the chemicaJ structures miceweregivenCTX(0.1p-g/kgofbodyweight
determined (Murata et al.,1989). Among them, Group 3: Fifteen mice were given low CTX
CTX is the mostpotent.A feature ofciguatera is (0.05fjLg/kg). Micein groups 2 and 3 were given
its obstinacy and recurrence of attacks (Bae- the phyeotoxin in a similar manner to group 1.
nis,1968,Ba'gnisctaL 1979). Ourstudyofshort- Two mice each from group 2 were sacrificed by
term, successive administration ofCTXata low cervicaldislocation5hoursaftertheintubationat
dose,confirmedthatCTXhasacumulativeeffect the 12th, 14th, ISth and 23rd week from the
on the cardiac tissue. In experiments reported beginning. Afterthetreatmentallsurvivingmice
here we re-examined the potency ofthe cumula- were fed a standard diet (CE-2.Nihon Clea Inc.,
tiveeffectsofthetoxinon themousehearttissue. Tokyo). Two mice from the group 3 were also
killedand controlled in a similarmannertomice
MATERIALSAND METHODS in group 2 at the 1Sth and 40th week.
Toxin
CTX used herein was isolated from reefsnap- MorphologicalExamination
pers {Lutjanus bohlar) from Micronesia and
Afternecropsyall internal organswerefixedin
Okinawa. Phyeotoxin was purified as described 10% neutral formalin and embedded in paraffin.
bmyinLiemgarlalnedtheatladl.os(e19o8f9)C.TOXne24mhorussaeftuenriti pis atdh-e TanhdePslAiSd.esFfoorr lTiEghMt.mipcirecoesscoopfythweerheearstt,aiknieddneHyE.
ministration into 20gmouse and is equivalentto and liver were fixed by cold paraformaldebyde-
0.35p-g/kgbodyweight(Yasumotopcrs.comm. i glutaxuidehydesolution(finalconcentration:2%)
OralLD50was notdetermined becausethe avail- and immersed in buffered 1% OSO4 at room
abledose ofphyeotoxin was limited. temperature. Then dehydrated in a series of
graded ethanol, embedded in Epon 812.and cut
ExperimentalAnimals withadiamondknifeonaPorterllultratome.The
Male ICR mice (4 weeks ol age weighing 20- ullrathin sections were stained with urany]
622 MEMOIRS OFTHEQUEENSLANDMUSEUM
W
-w w* v
/ - te v litJ:v*
!
FIG. A,Electron micrographofheartfrommouse given 0.1p-g/kgCTXorally byintubationsonceaweekfor
1.
25weeks.Betweencardiacmuscles(M)arethickbundlesofcollagen(Co). Bloodcapillaries(C)areembedded
in the collagen fibers. V: vein. Bar: 10u,m. B, Electron micrograph ofthe heart from mouse given O.lpg/kg
CTXorallybyintubationsonceaweekfor25weeks.Inasmallvein(V)athrombus(arrowheads)isdeveloping.
M:cardiacmuscle.Bar: lOpjn.C, Electron micrographoftheheartfromamouse given0.05 pg/kgCTXorally
by intubation for40weeks. Nodiscerniblechanges areseen.M: cardiacmuscles. Bar: 10p.m.
PATHOLOGICAL CHANGES IN MURINEHEARTSBYCIGUATOXIN 623
acetate and lead dime and examined with a markedly swollen and ruptured with seventy of
Hitachi H700HTEM. changedependenton dose. Theseedema may be
causedby theincreasedinfluxofNa*channelsof
RESULTS cardiac musclecells (Ohizumi l990). Inourpre-
1
vious report, short-term successive administra-
Oral administration of CTX at a dose of tion ofCTX at low dose resulted in acumulative
0.1|ig/kgresultedin noabnormality,whereas i.p. effect of CTX on the mouse heart (Terao et al,
injection ofthe same dose caused severe watery 1992). In the present study, an intermittent dose
diarrhoea within 10 minutes. In contrast, mice ited in similarsevere morphologicalchanges
injected i.p. with the phycotoxin at a dose of in theheart tissue. CTX may bind very tighti
0.05|xg/Vg induced no diarrhoea at all. Na* channels on the cardiac muscle cells or to
Even at the ultrastructural level a single oral those on theendotheliumofthe capillaries m the
dose of0.1fcg/kg ofCTX producedno abnormal interstitium.
changesintheheartmuscle. Long-term,intermit-
tentadministrationofCTXatadoseof0.05p-g/kg L1TF.RATURF CITED
induced nopathological changes in the heart tis-
sue until 40 weeks. En contrast, intermittent ad- BADEN, D.G. 1983. Marine food-homedinoflagellate
ministration of 0.1M-g/ke CTX once a week for toxins.Pp 99 150.JnG.H.Bourne&J.F.Daniel!i
over 12 weeks resulted in severe morphological \ais) International Review of Cytology 62*
changes in heart tissue. (Academic Press: New York).
Mice given 0-05u..g/kg CTX showed no abnor- BAGNIpoSi,soRn.ing1]96m8.FCrleinnicchalPoalsypneecstisa.ofHacwiaguiaiteMreadi{fciaslh
mal behaviour through the experiment. Mice
treated with CTX at an intermittent oral dose of BAl;NJIoSur.naR.l,2K8;UB25E-R2S8.Kd, T. & LAUGIER, S. 1979.
O.Iftg/kg also showednoabnormalityduring the Clinical observations on 3,009cases ofciguatera
first 12 weeks. After that, however, shock often (fish poisoning'i in the South Pacific. ATOC
Occurred shortly after administration of CTX. Journal of Tropical Medicine and Hygin<
Usually die animals recovered spontaneously tOU7-1073,
within 10 minutes. After 18 doses or at the 18th LEGRAND. A.M., UTAUDON, M., GENTHON,
week two mice were sacrificed. Both ventricles IN.. BAGNIS, R. ft YASUMOTO, T 19K9.
of the animals were dilated at necropsy. His- ., tnd some propertiesofCTX, Journalof
topathoiogically, multiple single cell necroses Applied Physiology 1: 183-188.
were often seen in the mural orpapillary muscles MUKATA, M. LEGRAND.A.M., ISHlfiASHI, Y. &
of the left ventricle. TEM examination showed YASUMOTO,T 1989.StructuresofCTX andits
swelling ofmyocardiac cellsandedemabetween coogener. journal ofAmericanChemical Society
111 8929-8931.
becleuelnlcdstlreobsne-odcfeanmmsuees.clOrceocufanisbdireoedsn.alaMindtdiossctohhcoeinadtmriaiotnaroiifnxitnhwteeasrse- OHIZUo]f9M8CI1,T.XMYo.i,dnetShHoefJgaBuiAenxTecAait.apitoSgr.vya&asnddTefAienCrheiHnb1si.tBoJAroyNuarAcn,tailoKon.sf
calated discs was noted. Blood capillaries were Pharmacology and Experimental Therapeutics
embedded by bundles of collagen fibers and 221: 748-752.
electron-dense flocculent materials (Fig,]A), TERAO, K.. fTO, E St YASUMOTO, T. 1990.
Capillaries and small veins were often occluded Paihoniorpholngieal studies on experimental
by accumulation ofblood platelets (Fig.IB). nuatoKwicoMs and ciguatoxicosis in mice. Pp.
Mice given 05u.g/kg CTX produced nochan- T0\ In TJ. Tostexon fed.), 'Ciguatera Puerto
ges in heart tissue until 40 weeks (Fig.1C). Rk» 1990* (Polyscience Publication: Quebec).
TERAO. K ITO, E. OARADA. M., ISHlfiASHI. Y.,
DISCUSSION LEGRA, ND, A.M. & YASUMOTO. T. 1991
Light and electron microscopic studies of
1 ologic changes induced in mice hy CTX
The most prominent morphological changes poisoning. Toxkon 29. 633-643.
afteradministrationofCTXoccurredin the hcaii TER vo. K ITO, E. & YASUMOTO, T. 199? Light
muscles (Terao et al.,1990,199IT992) Almost and el,ectron microscopic studies of the marine
all cardiac muscle cells and the endothelium of heart after repeated administrations of CTX or
blood capillaries in the cardiac mterstitium were CTX-4c. Natural Toxins I; 19-26.
