Table Of ContentTHE MOUSECiGUATOXIN BIOASSAY: DIRECTIONS FOR USETO CONTROL FISH
TORCONSUMPTION
JEAN-PAULVERNOUX
Vernoux,J.P. 1994OS01:Themouseciguatoxinbioassay:direction*Rh usetocontrolfish
for consumption. Memoirs ofthe Queensland Museum Mi's\: 625-629 Brisbane. ISSN
0079-8835.
Ciguatera fishpoisoningcausesseriou*health problems amund theworld but the diversity
and heterogeneity of ciguatoxins arc delaying chemical and immunological remedies.
Realisticmethodslorciguatoxin screening offish are needed torpublic health studies. The
mouse bioassay may prove useful since it is simple ami relatively cheap. Qualitative and
semi-quantitative analyses for ciguatoxins from 50, 100 Of 200g i>l lish tissue using
respectively 2(6or 12animalsare precisely described.
Jean-Paul Vernoux, LaboratoryofCellularandMolecuhirPhvsiolow. UniversitvofCaen,
Esplanadedela Pali - 14032 CAENCedex, France 2 February 1994.
Ciguateraisahumanillness,sporadicinnature, per g of flesh). So only trace quantities of
caused by the ingestion of a wide variety offish ciguatoxinsareneeded to elicithumanpoisoning
typically associated with coral reefs. These fish (0.25ppb). Therefore only those detection
are transvectors for multiple ciguatera toxins methods capable of detecting Lis little a^ 250-
(mainly ciguatoxins) acquired through theirdiet 500pg of CTX per gram of fish flesh need be
(Vernoux & AbbadelAndaloussi.1986;Legrand considered. HPLC and immunological methods
etal.,1990;Lewts&SelJin,1992). Fishpoisoning could be appropriate methods. Unfortunately,
is present in many coral reefareas of the world until now the detection of these extremely low
(Bagnis,1981> with 10,000-50,000 persons af- levels of multiple ciguatoxins (>20, Legrand et
fectedeachyear. Itsincidencehasbeenestimated al.,1990) has delayed the use ofHPLC methods.
at 1^/000 population in the Pacific area (Bag- Immunological methodsthat possess thedesired
nis,1979; Lewis.1984; Yasumoto et aLl sensitivity and specificity have already been
4.2/000in the Virgin Islands (Olsen et al-,1984), developed (Hok.ima.1990; Park et al.,1992).
and 3-10/000 at Saint Barthelcmy Island (Ver- However,thepotentialofthesemethodsforlarge
noux, this memoir) and in the Saintes Islands scale use remains lo be demonstrated, at least in
o(fCzaeirrntriacvhelo,wtertoapl.ic,a1l98r4e)e.fWfiisthhotrhecionncsruemaesirnsg(uisne- pciagrutatdouxienstointfhiesh pcoosnstiabmiilniattiioens.oAf bmiuollotgiipclael
habitants or tourists) are constantly moving and assay whichencompasses all thedifferenttoxins
ciguatera is being documented in temperate amnedthgoidvesfotrotpalubtloixcicihteyalctohulcdonbteroulseodfafisisah.roTbwuoM
regions (Lange et al.,1992). Thus, ciguatera is a
biCMtesays having the desired sensitivity arc Ihc
pwroarcltdicahleaslctrheepnrionbglemmetahnoddtfhoerreciigsuaatenreaedtoxfionrs.a uiosquito injection bioassay (Chungue ei al,
19*4) and the mouse injection bioassay (Hof-
hAemaoluthntpsroobflecimgauxaetovxeirnylinowf.isOhntehaatppporsoeacahptuobltihce fismmaonreetcaoln.v,e1n98i3e)n.t,Thsiempllatet,erspiescpirfeifcearnrdedhassinbceeenil
estimation of dangerous levels of toxin in fish widelyused.Afterhavinguseditfor20years, wc
tissue is to dose it in those portions that had present some recent developments in its use as
elicited human ciguatera poisoning. Some screeningmethod.
authors did this using mouse or mosquito bioas-
says(Yasumotoetah.1984;Chungueetal. 1984; METHODS
Bagnis et 31.4987; Vernoux, this memoir). As-
suming that 50Ong CTX can kill cJOOOg of
mouse li.p. LDsoofCTX into micc=0.45u,g/kg), Rlesh Extraction
these studies show that the minimum threshold Raw orcooked minced flesh can be usedsince
for the human pathogenic dose is c.50-100ng. ciguatoxins arc heat resistant. If cooking, use a
This level corresponds to 10Q-200g of mouse boilable cooking pouch filled with raw minced
killed by a 200g fish portion i.e. the flesh sample sample and boil in waterfor30 minutes.
hasa specific toxicity nf0.5-1g ofmouse killed A typical procedure to extract 50g of tie-
, 1
626 MEMOIRSOFTHEQUEENSLANDMUSEUM
TABLE 1. Testingoffish forconsumptionby the mouse bioassay
1 prepareLRfor50goffleshanddilutewith 2ml of1%Tween60salineat37°Cand homogenizethoroughly.
2, inject i.p. into 2 male mice (18-24g) at dosage d=0.04ml/g ofmice (i.e. lg equivalent offlesh per gram of
mouse).
3,Observesymptomsduring4hrandconcludeforciguatoxinpresence(=penileerection)forneurotoxinpresence
(=respiratory distress) or for okadaic acid or fatty acid presence (crawling gait, slow breathing and general
cyanosis).
4, notedeath after24hrsand weigh the survivors. Usethe followingtableto indicateedibility.
Observedmortalityaftera Toxinconcentrationinflesh Lossofweightat24hr(>5%) Interpretation
lg.eq.offleshinjection/g
ofmouse
2/2 > lMUg/g.e.f - notedible
1/2 0.5to 1 MU^.ei yes notedible
0/2 <0.5MUg/g.e.f yes bordeline
no edible
1, homogenise 3 min in a Waring blender with etc. Note that the total weight of mouse to be
150ml ofacetone, 2, filterontoabuckner funnel injectedrequires atotal volumebelowthatavail-
and wash the remaining cake with 30ml of 80% able in the experiment and the use ofmice <20g
acetone; discard the cake, 3, remove acetone on forthe highestdosage may be necessary.
a rotary evaporator under reduced pressure and Doses <lg.e.f/g are injected in 0.8ml per 20g
reducethevolumeoftheremainingaqueoussolu- mouse by carrying out dilutions directly in the
tion to 30ml (add waterifbelow), 4, add 10ml of syringe with 1% Tween 60 saline at 37°C. The
ethanol, shake and extract twice with 40ml of approach described above allows determination
diethylether,5,removediethyletherandresidual oftheLD50and the minimumlethal dose (MLD)
waterunderreducedpressure(additionofethanol which is the lowest dose capable of killing two
allows to remove quickly residual water), 6, dis- mice in the two mice group (orone mouse in the
solve the diethyl ether residue in 25ml of 80% one-mouse group) after 24 hr. The toxin content
methanol and wash twice with 50ml ofhexane; isexpressedintermsofMouseUnitsgram(MUg)
discard the hexane solubles, 7, remove methanol where IMUg is lg ofmouse killed by the MLD
and water under reduced pressure; resulting dry (ortheLD50) expressed in g.e.f/g. Thetoxincon-
residue is called lipid-soluble residue (LR), 8, if centration isexpressed in MUgpergramofflesh
weight ofLR is >75mg dissolve in 10ml of80% (MUg/g.e.f) which is the reciprocal ofMLD (or
methanol and wash again twice with 20ml of LD50).
hexane, 9, emulsify LR in 2ml of 1% Tween 60 The suitability offish forconsumption may be
saline and keep at-20°C until use. controlled by mouse bioassay using 50g offlesh
(Table 1). Quantitative and semi-quantitative
DeterminationofToxin Concentration conclusionsarepresented.Twohoursareneeded
LR emulsified in 1% Tween 60 saline was toprepareLR.IfdissolutionofLRisdifficult,0.
hweeaitgehdinagt 3178°-C24agnd(2i.mpi.cienjpeecrteddosien)t.oAmasleerimesicoef msolluotfioent.hAanonlegcaatnivbeecoandtdreoldipserrun1.us9imnlgotfwTowmeiecne
doses that vary in a geometric progression by a injectedwithablanksolution(withoutLR).Non-
factorof1.1938areassayed.Dosesareexpressed toxic fish extracts give negative results (they
in gram equivalents offlesh per gram ofanimal elicit no symptoms).
(g.e.f/g). They were chosen in succession in the Acute toxicity may be determined in two steps
following series of numbers running from 10""1 (Table 2) with a minimum ofanimals (Lorke et
to 10" which increase successivelyby a constant al.,1983). For this method 200g of flesh should
powerof 10 (since 1.193813 x 10""1 = 10"). Four- beextractedandfourhoursareneededtoprepare
teennumbersinsuchaseriesare0.10,0.12,0.14, theLR.Intheinitialinvestigation,whichrequires
0.17,0.20,0.24,0.29,0.35,0.41,0.49,0.59,0.70, anamountofextractcorrespondingtoabout lOOg
0.84, 1.0. Onegramequivalent corresponds here of flesh and 6 mice, an approximate range of
to 0.04 ml ofLR solution and the volumes to be doses producing the toxic effects is established.
injected are respectively: 0.04 ml/g of mouse Normally this initial investigation wouldinclude
multipliedbythenumbers indicatedin this series doses used inthecontrol offish forconsumption
i.e. 0.004ml/g;0.0048 ml;0.0056ml;0.0068 ml, method i.e. injection of 1g.e.f/g as afirst step; in
MOUSECIGUATOXIN BIOASSAY 627
TABLE2. Determination oftheacutetoxicity ofLR,prepared from200gofflesh, in twosteps.
1ststep EXPERIMENTAL 2ndstep RESULTS
Dosesdi in g.e.f./gofmouse Approximate Dosesd2ing.e.f./g Correspondingtoxin
Letha(l5itpyo3ssiinbitlhietifeisrs)ttest dceodnucecnetdrattoixoinn chos(e2nmfiocrethpeersedcoosne)dtest coanccecnotrrdaitnigotnoitnhMeU(g/MgL.De).f.
inMUg/g.e.f.
lc 0.49 0.24
m
0/2 0/2 0/2 <] h 1.7 ItAf 0.5 0.59 0.71 -
(2.0) (1.7) (1.4)
1/2 0/2 0/2 >0.5and<l 1.4 1.2 [0.84] 0.71 0.83 -
(1-4) (1.2)
2/2 or 1/2 0/2 >1 and<2 0.84 0.7 0.59 1.00 1.19 1.43 1.70
(1.0) (0.84) (0.70) (0.59)
2/2 2/2 or 1/2 >2and<4 0.41 0.35 0.29 2.04 2.44 2.86 3.45
(0.49) (0.41) (0.35) (0.29)
2/2 2/2 2/2 >4 0.20 0.17 0.14 0.12 4.17 5 5.88 7.14
(0.24) (0.20) (0.17) (0.14)
aNumberofanimalsdied/numberofanimalsused
bOnemouseperdose
cThisdoseallowscontrol offishforconsumption.
[ ] Possibleonly when control offish forconsumptionis used insteadofthe firsttest.
that case according to the observed lethality 0/2 the procedure with acetone (Vernoux,1981) and
or 1/2 it may be possible to bypass the 0.49 and we havebeen using this method since 1981.
0.24g.e.f/ginjections (butnotifa2/2 lethality is In our method the LR yield must be below
observed).Basedontheseresults,furtherspecific 0.15% of the flesh since the less impurities
doses are administered to a group ofone mouse present the more marked the symptoms in mice
ortwoperdosedependingonthepredictedtoxin foragivendose.Dosesreceivedbymicewiththis
concentration (<1 or >1MUg/g.e.f). From the method do notexceed 1.5 mg ofLR/g ofmouse.
resultsofthesetwotests,6or7 successive levels Our proposed interpretation ofsymptomatology
ofdosage (di + d2) are then assayed. For a two- and lethality includes:
mice group an LD50 can be calculated by the -theuniquepropensityofciguatoxin to induce
method ofWeil (1952) using the equation penile symptoms i.epenile cyanosis and/ortran-
log LD50= log Da+ log R (f+ 1) sitory and incomplete erection (sometimes even
Da being the lower dosage level, R the reaching priapism i.e. complete and permanent
geometric factor and f a value given in Tables. erection seen following sub-lethal doses (Ver-
Thispermitsasimpleandrapidestimation ofthe noux & Bagnis,1976; Vernoux et al.,1985). This
LD50 with acorresponding confidence interval. symptom wasrecentlyconfirmedbyTeraoetal.,
For the one animal group per dose, LD50 is (1991)whopointedoutthepenisasatargetorgan
estimated as the geometric mean ofthedoses for forciguatoxin.
which 0/1 and 1/1 are found (Lorke et al.,1983). - the symptoms in mice after i.p injection of
&
The minimum lethal dose can be used instead of okadaic acid (Vernoux Moulin,1989) or fatty
LD50. acids (Vernoux,1981) are different from that
elicited by CTX but resemble the effects of
RESULTS ANDDISCUSSION maitotoxin, a toxin never detected in fish flesh
(Yasumotoetal.,1984).
- the existence ofa narrow range ofdoses (d -
ControlofFishfor Consumption 2d) between 0% and 100% lethality (Hoffman et
The proposed acetone method is much more al.,1983; Lewis & Endean 1984; Vernoux &
economic and is as rapid as the method ofLeeet Moulin,1989).
al. (1987) who used methanol as the extracting - the general observation of a minimum
agentinsteadofacetone forokadaicacid, atoxin pathogenic dosage only 1/4 to 1/3 the LD50
chromatographically related to ciguatoxin. The dosage and the link between the pathogenic
methanol method is convenientonly ifthe tissue dosage and loss ofweight (Chungue et al.,1984;
portions to be extracted are <10g. So we prefer Vernoux,1988).
628 MEMOIRS OFTHEQUEENSLANDMUSEUM
- additional observations suggest a 5 MUg or determined from the curve of dose (d) versus
10 MUg dosage/g of mice if survival time is survival time (t) particularly since the testcanbe
respectively about 1 hour and halfan hour but it conducted in one day. Nevertheless this method
may vary considerably with fish species (Ver- is convenient only if the toxin concentration in
noux andTahla,1989). Therelationshipbetween fleshis>2MUg,thuslimitingitsapplication.The
the mouse response and the quantity of toxin relationshipisd=LD50(1 + l/t)bord/LD50=(1
present is given in Table 1 and 2. + l/t)b i.e. number of MUg/g.e.f = (1 + l/t)\
Unfortunately b is fish-species dependent and it
AcuteToxicityDetermination varies from 2 to 3 (Vernoux,1991) thus com-
plicating the situation. So this method is of
Extracting200gisconvenientforinvestigating limited interest in controlling fish for consump-
fishforconsumptionandforquantifyingthetoxin
tion.
concentration in the 0.5-7.14MUg/g.e.f range.
This range is sufficiently wide to include toxin CONCLUSIONS
concentrations found in fish in the Australia,
Pacific area or the Caribbean. There is no upper
Presence of multiple ciguatoxins in fish flesh
lsiimnicteftohrethmeodreetetromxiincattihoenfolfesthoxtihnecolnescsenRtrLatcioonn-, led us to propose here a simplified mouse
ciguatoxin bioassay. Exhaustive description of
csounmterodl ionftfhieshtefsotr. cUonnlsiukmepttihoen,meatchuoted tfooxrictihtey ftihsehmbeytahnoydunsshpoeuclidalailsleodwhyitgitoenbeelaubsoerattoorcyo.ntTrhoel
determination takes more than one day to con-
duct.Fortunately,stabilityoftoxinsin 1%Tween lviemniiteendt qfuorantinivteystoifgaftliensghfuisshedfo(r20c0ogn)suimsptcioonn-
-6020s°aClinfeorisupcotmop6lemtoentwhhse.n samples are stored at and itallows to determine toxin concentration in
ThegeometricfactorR= 1.1938waschosento atlhlesmiutlutaitpiolnes.moTuhseefciixgeudatmoextihnobdiosahsosualydmeretphloadcse
provide a closely spaced series ofdosage levels. for which toxicological bases are not very clear.
Thisgeometricalseriesofnumbersincreasessuc- Standardisation of the mouse strain could be
cessively by a constant power of 10 as already realised with a known toxin having a similar
mentioned above. Furthermore, as (1.1938)4 = physiologicaleffect,brevetoxinforexample. We
2.0 another geometric factor R = 2 can be used hope that our proposals will gain wide accep-
and numbers of the corresponding series are tance, since the mouse ciguatoxin bioassay
therefore included in the first one. The two-step proposed here provides both a qualitative and
method shown in Table 2 was developed using semi-quantitative bioassay for ciguatoxins in
theseseries. SincethefirstoneisacloselyspMaLceDd fish.
series, this enhances the precision of the
determination. In this case we observed that the ACKNOWLEDGEMENT
MLD
values obtained with two animal groups
wereequivalenttotheLD50valuesobtainedwith This study was realised within the scope ofthe
four animal groups (Vernoux and Tahla,1989).
FrenchNationalProgramonMarinePhycotoxins
This experimental correlation can be easily ex-
and supported by an Tfremercontract.
plainedsincetheslopeofthedoseresponsecurve
for ciguatoxin is high with a narrow dose range LITERATURECITED
(d-2d) between 0% and 100% lethality, thus in-
MLD
cluding and LD50 values (Vernoux and BAGNIS, R. 1979. L'ichlyosarcotoxisme de type
Moulin,1989). Nevertheless the two-mice group ciguatera Phenomene complexe de biologie
orevenone-mousegroupalsogivereliable LD50 marine et h:umaine. Oeeanologica Acta 4: 375-
values (Weil,1952; Lorke,1983). Here with the 387.
two-mice group, to calculate LD50 we use the BAGNIS, R. 1981. Etude morphologique, biologique,
methodofWeil (1952) sinceitiseasierand more toxicologiqueetecologiquederagcntcausalprin-
rapid than the method ofLitchfield & Wilcoxon cepsdelaciguatera, lePeridinienGambierdiscus
(1949) and the former approach allows the con- toxicus.Thesed'EtatenBiologieHumaine, 180p.
fidenceMiLntDerval to be estimated. However, we BAGNIUnSi,verRs,ityBAdeRSBIorNdAeSa,uxMI.I,, FPraRnIcEe.UR, C, POM-
prefer determination to LD50 calculations PON, A., CHUNGUE, E. & LEGRAND, A.M.
since a greateraccuracy is not necessary in view 1987.Theuseofmosquitobioassayfordetermin-
oftherangeofvariationfrMomLoDnedosetoanother. ingthetoxicitytomanofciguateric fish. Biologi-
Itmightbethoughtthat orLD50could be cal Bulletin 172: 137-143.
MOUSECIGUATOXIN BIOASSAY
f>2^
CHUNGUE, E., BAGNIS, R. & PARC, F. 1984. The munobead assay forthedetectionofokadaicacid
use of mosquitoes (Aedes atgypH} to detect and related DSP toxins in shellfish. Presented at
ciguatoxininsurgeonfish(Ctenorhaetuxxtriatus) 2nd Internationalconferenceonshellfishpurifica-
Toxicon22: 161-164. tion. Rennes (France). April 1992.
CZERNICHOW, P., DROY. J.M., EZELIN, F. & TERAO, K.. ITO, E., ORADA, M.. 1SHIBASH1, Y.,
LEROY, J. 1984. La ciguatera aux lies Saintes LEGRAND. A.M. & YASUMOTO, T. 1991.
(Guadeloupe) inaladie iransmise par les pois- Light and electron microscopic studies ot
.
sons, Lapresse M£dicale 13; 222 pathologicchangesinducedinmicebyciguatoxin
HOFFMAN, PA.,GRANADE. H.R. & MCMILLAN, poisoning.Toxicon29; 633-643.
J.P. 1983.Themouseciguatoxinbioassay: adose VERNOUX,J.P.&BAGNIS.R. 1976.Fracoonnemcnt
response curve and symptomatology analysis. d*cxtraits lipidiques ciguaioxiques en milieu al-
Toxicon21: 363-369. exin. Biochimie 58: 479-484.
HOKAMA, Y. 1990. Simplified solid-phase im- VERNOUX. J.P. 1981. L'ichtyosarcotoxisme de type
nuinobead assay lor detection of ciguatoxin and ciguatera aux Antilles cten Polyn^sie Francaise:
relatedpolyethers.JournalofClinical Laboratory de crgiiatoxicitd CI en'ainc trophique
LANGAEn,alWy.sRi.s,4S:N23Y1D-E21R7,.FR &FUDALA.PJ. Im92. dCfetaguuaxteIr.igene. PhD Thesis, University of Bor-
Travel and ciguatera fish poisoning. Archie VERNOUX. IP. LAHLOU. N,. ABBAD EL AN-
LEE.JI.nSt.e,rYnaAlNMeAdGiIc,inTe.,1K5E2:N2M0A4,9-R2.05&3.YASUMOTO. DPhA.LO19U8S5.S1A,sStumdyR1ofYEthCeHcEi,guNa.to&xinMiAnGiRndAiSvi,duaLl.
T. 1987. Fluorimelric determination ofdiarrhctic Caribbean fish. ActaTropica 42: 225-233.
shellfish toxins by high performance liquid VERNOUX, & ABBAD EL ANDALOUSSI,
J.P. S.
chromatography. Agricultural and Biological
1986. Heterogeneity of ciguatoxins extracted
LEGRMYCAhAUeNmSRDiUsA,tMTrOyAA"TM,5O,1,: 87MCT.7R.—U81C899H101.F.STHC,IhrBPoAhmSatHBoIgA,rGaNpIhYiS.c, aRn&.d, VERNBBFOiraoUorcmXihfh,iicmsLhilecPeam6u8y1g:9:h8t288.aa7ts-Ltp2haee9cc1cit.ogsausattcoeprfiatdhdecamFnrsteoPnliclohegAdineqtiuSlaeliessn..t
cspiegcutartaelraetvoixdiensn.cePp.to3r74th-e37p8r.eIsnenEc.eGroafnemluilcttipalLe, tQKtcofogjques el preventifs. Oceanolouica Auj
11:37-46.
Toxic marine phytoplankton* (Elsevier VERNOUX, J.P. & MOULIN, F. 1989. Intoxications
Netherlar:.
LEWIS, NX). 1984 Ciguatera in the the Pacific: in- alimentaires a dinoflagelicsci dosage (Jes toxines
cidence and implications for marine resource assoeieesauxintoxicationsdetypeeigualeriqueet
development. American Chemical Society, Sym- diarrheique dues a la consommation d'animaux
posiumSeries 262: 289-306 mariflS Science des Aliments, hors seYie III 9;
LEWISLh.cRf.lJe.sh&anEdNvDisEceArNa orfR.the19b8a4r.raCciugduaatSopXhtxnra&eWnWa VERM68j-U83X.,J P.&TAHLA,F. 1989.Fractionationand
LEWISle,flRo.,JT.o&xiScEoLnL2I2N:,8M0.6-14992.Multipleciguamxins pciugruiaftiocxaitnisonexotrfacstoedmefrommusCcaurliabrbeaanndfisvh.isCcoerma-l
in the fleshoffish. Toxicon30: 915-919. parativeBiochemistry andPhysioloey94F-L 499
LITCHFIELD. J TJr & W1LCOXON, F. 1949. A 504.
simplified method of evaluating dose effect ex- VERNOUX, LP. 1991. Moyens d'mvestigation pour
periments. Journal of Pharmacology and Ex- confirmer une intoxication de type ciguatenquc.
perimental Therapeutics 92: 99-1 13 InFremy,J.(ed.h 'ProceedingsofSymposiumon
LORKE, D. 1983. A new approach to practical acute marinebiotoxins/ (CNEVA).
toxicity testing ArchivesofToxicology 54:275- WE1L.CS. 1952. Tables forconvenientcalculation ul
287. Dilian-effective close (CDso) and instructions in
OLSEN, DA., NELLLS, D.W. & WOOD, R.S. 1984. fheir use. Biometrics 8: 249-263.
Ciguatera in the Eastern Caribbean. Marine YASUMOTO, T.. RAJ, U„ & BAGNIS R. 19M4
Fisheries Review 46: 13-18. Seafood poisoning in tropical regions'.
PARK. D.L.. FREMY. JM, GAMBOA, P.M. & •ratoryofFoodHygiene.FacultyofAgricul-
GLEIZES, E. 1992. Innovative solid-phase im- ture,Tohoku University: Sendai). 74p.
