Response of Mice to Gambierdiscus toxicus Toxin

BONNIE A. KELLEY, DAVID J. JOLLOW, EDWIN 1. FELTON,MICHAEL S. VOEGTLlNE, and THOMAS B. HIGERD

Introduction

Ciguatera seafood poisoning is a seri­ous human illness brought on by ingest­ing certain coral reef-associated fish intropical and subtropical regions. Thetoxin carried by these fish was firstisolated by Scheuer et al. (1967). Thepresence of the toxin has been con­firmed in red snapper, Lutjanus bohor,moray eel, Gymnothorax javanicus,amberjack, Seriola aureovittata, andothers. Toxic fish probably acquireciguatoxin through their diet, and thatone source of toxin is probably the ben­thic dinoflagellate, Gambierdiscus tox­icus, found in association with certainmacroalgae of coral reefs (Yasumoto etal., ICJ77). Extracts of laboratory growncultures of G. toxicus, however, haveyielded a more polar toxin similar tomaitotoxin described from surgeonfish,Acanthurus sp. (Yasumoto et aI., 1CJ79).The relationship of this dinoflagellatetoxin to ciguatoxin is unknown, thoughone possible explanation is that the dino­flagellate toxin becomes chemicallyconverted as it is metabolized duringtransfer through the marine food web.

ABSIRACF-Response to toxins extractedfrom cultured Gambierdiscus toxicus wasevaluated in mice. Toxin preparations ad­ministered intraperitoneally or intravenouslygave similar dose-response curves, whereasoral administration elicited no response.Time-to-death determination was dosedependent and was quantitated to the dose­response based on lethality. The 48-hourlethality dose curves for the dinoflagellatetoxin were comparable to those previouslypublished for ciguatoxin extracted from fish,whereas the time-to-death curves showed astrong difference. The W so response ofscheduled multiple injections suggested a 4­to 8-hour half-life for toxin activity in themouse model. Sex and strain of the mousedid not affect susceptibility.

48(4), 1986

Toxin(s) extracted from cultured G.toxicus injected intraperitoneally (i.p.)into laboratory mice is reported to evokegross symptoms indistinguishable fromthose reported for partially purified fishextracts containing ciguatoxin (Hoffmanet aI., 1983; Sawyer et aI., 1984). Bothtoxins exhibit similar dose-responsecurves and both elicit in mice a strik­ing hypothermia, which is reversed byincreasing ambient temperature (Sawyeret al., 1984). It is important, therefore,that the biological and chemical rela­tionship between these toxins be clari­fied. In this investigation, we haveutilized the mouse bioassay to examinefurther the biological activities of thedinoflagellate toxin for comparison withciguatoxin and to gain an estimation ofthe biological half-life of G. toxicustoxin in mice.

Materials and Methods

Cells of G. toxicus, Adachi and Fuku­ya, were supplied by Rick York of theHawaii Institute of Marine Biology. Theisolate, clone T-39, was cultured in100-liter vats in F/2 medium supple­mented with a seaweed extract. Har­vested dinoflagellate cells were shippedto South Carolina in aqueous methanol,and upon arrival, cells were extractedfor 7 days at room temperature in meth­anol:water (80:20). The suspension was

Bonnie A. Kelley is with the Department ofBiology, Pembroke State University, Pembroke,NC 28372; David 1. Jollow is with the Departmentof Pharmacology, Medical University of SouthCarolina, Charleston, SC 29425; Edward T. Feltonand Michael S. Voegtline are with the Departmentof Basic and Clinical Immunology and Micro­biology, Medical University of South Carolina,Charleston, SC 29425; and Thomas B Higerd isalso with the Department of Basic and ClinicalImmunology and with the Charleston Laboratory,NMFS Southeast Fisheries Center, p.o. Box 12607,Charleston, SC 29425. This paper is Publicationno. 775 from the Department of Basic and ClinicalImmunology and Microbiology, Medical Univer­sity of South Carolina.

clarified by centrifugation, the super­natant dried, and the resulting solidsweighed and dissolved in absolute meth­anol. This suspension was filtered,designated as crude dinoflagellate ex­tract, and stored at 4°C.

Assays for toxicity were conducted onICR female, ICR male, and C57BL/6female mice all weighing approximate­ly 20 g each. Animals were maintainedon Wayne Laboratory Animal diets(Lab-Blox) 1 and water, ad libitum. Aknown quantity of the crude dinoflagel­late extract was resuspended in phos­phate-buffered saline (PBS) containing5 percent Tween-80 and administered(in 0.2 rnl aliquots unless otherwisestated) to mice intraperitoneally, intra­venously (Lv.), and by gavage. Controlanimals received an equal volume of thesolvent vehicle. Lethality was recordedat 48 hours for some studies, and time­to-death after injection was recorded forother studies. One mouse unit (MU) oftoxicity is defined as that quantity ofcrude extract capable of eliciting a fataldose in half of the test animals by 48hours. Body temperatures were re­corded using a YSI rectal probe (Model43TA, Yellow Springs Instrument Co.,Yellow Springs, OH) within 2 hoursafter administration to obtain early in­dications of toxicity.

For the retention time study, a stocksolution of dinoflagellate extract (13.3MU/rnl) was prepared in PBS contain­ing 5 percent Tween-80 (67 ~ extract/rnI). Each ICR female mouse was ad­ministered 0.15 rnl (10 JAg) i.p. of thestock solution or 0.15 rnl of a 1:3 dilu­tion; a reduced volume was used due toadministration of multiple injections.One control group received 10 JAg of tox-

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35

Table 1.-The effect of administration route on lethal­ity of G. toxlcus extract.

Adminis- No. of Time totration ani- Percent deathroute Amount rnals' response2 (h)

I.p. 50 ~g 6 100 4to 22

I.v. 50 ~g 6 100 1 to 3

Gavage 100 ~g 4 a

'Female ICR mice.'Percentage of fatalities after 48 hours.

•

•

•

0.1 0.2 0.4 1 2

G. toxicus extract (mg/kg)

ICR, male

ICR, female

C571BL. female

Figure I.-Percent response (death) in48 hours displayed by three mousestrains to i.p. administration of G. tox­icus extract diluted in PBS. Datapoints represent a minimum of 8animals.

8.04

50

50

100

100

100

ship between the dose of toxin and time­to-death in the mouse. For comparisonwith these published reports, we ob­served the lethality of the dinoflagellatetoxin as judged by time-to-dealth re­sponse in relation to the 48-hour LDsoresponse (Fig. 2). Groups of test micewere injected i.p. with dinoflagellate ex­tract equivalent to 2-33 MD. At thesedoses, death occurred between 2 and 16hours, and the mean time-to-death was

Determination ofMean Death Time

A standardized mouse bioassay hasbeen universally accepted to determinethe toxicity of saxitoxin, a marine toxinof dinoflagellate origin and the causitiveagent of paralytic shellfish poisoning.The method described by Schantz et al.(1957) involved determination of mediandeath time for a given dilution of a sus­pect extract injected i. p. relative to asaxitoxin standard. For ciguatoxin,Tachibana (1980) reported the relation-

2Baden, D. Department of Biochemistry, Univer­sity of Miami School of Medicine, Miami, FL33101. Personal commun., 1982.

death) than mice injected i.p. The tox­icokinetics of the dinoflagellate and fishtoxins are not known. It is interestingthat the time to develop overt symptoms,such as hypothermia in mice (Sawyer etal., 1984) or clinical symptoms in man(Bagnis et aI., 1979), occurs over aperiod of hours. In view of the high lipo­philicity of these toxins, which should ~

enhance their equilibration across mem- ~

branes and promote their accessibility ~ 50

to the site(s) of action, a much more gu

rapid response, perhaps within minutes, ~

would be expected.The lack of any response, including

temperature depression in mice admin­istered the dinoflagellate extract bygavage, raises interesting questions re­garding the toxin's rate of absorption,physical or biochemical inactivation,etc. Preliminary studies (not reported),in which the toxicity of the dinoflagel­late extract was not lost when incubatedunder acidic conditions or with variousmouse tissue extracts, suggest that sim­ple physical or enzymatic inactivationwas not responsible for abrogation ofdinoflagellate toxicity when adminis­tered orally. The observation that theroute of administration influenced thedose-dependence of the toxicity was firstmade by Baden2 and is in contrast tothe animal response with fish toxin(putative ciguatoxin) described by Ban­ner et al. (1960), in which an equiva­lent response was obtained whethermice were injected with toxic fish ex­tracts or fed the extract equivalent totwice the injected dose.

Results and Discussion

Sex and Strain Differences

Route of Administration

To determine the effect of the routeof administration on the toxicity of G.toxicus extract, animals received thetoxin in three ways: Intraperitoneally,intravenously, and orally (p.o.). Re­sponse of mice to 10 MU of toxin ad­ministered by i.p. and i.v. routes wasuniformly fatal (Table 1). Average time­to-death, however, was much shorterfollowing i.v. injections. Administrationby gavage of approximately 20 MU re­sulted in no test mice fatalities.

The potency of the dinoflagellate tox­in, as defined by percent fatalities of thetreated mouse populations at 48 hours,was not significantly different betweenmice receiving the dinoflagellate extracti.p. or i.v. However, mice administeredthe toxin i.v. responded earlier (time-to-

Determination of the dose-responserelationship to the crude dinoflagellateextract administered i.p. to ICR female,ICR male, and C57BL/6 female micesuggested that sex and strain of themouse did not markedly influence tox­icity (Fig. 1). The LDso's for ICR fe­male, ICR male, and C57BL/6 femalemice were estimated to be 260 ~g/kg,

393 ~glkg, and 192 ~glkg, respectively.ICR females were used in later tests.

The i.p. injection of ciguatoxin ex­tracts into laboratory mice has been areliable bioassay method for ciguatera­associated toxins and has been widelyadopted by investigators of this field.The variability that may be present inthe assay due to the sex and strain ofmice used has not been studied. Theresults of this limited study indicatedthat no apparent difference should beexpected in dose-response with respectto sex and strain of the test animal.

ic extract as a single injection, andanother control group received 3.3 ~g

in a single injection. Each test animalin the eight remaining groups receivedthree equally spaced injections over atime interval ranging from 0 to 30 hoursbetween individual injections. Lethalitywas recorded after 48 hours of the lastinjection.

36 Marine Fisheries Review

'6

~ 14

212 :

~1O!- ,~ 8 i~ 6 :

: 4 \

~ 2 \

o0':----:'='O-------:2l::-0-----:f:30,-------!40

Mouse units

Figure 2.-Relationship between G.toxicus extract dose and time-to-deathofICR female mice (solid line). Micereceived doses expressed in equiva­lents of mouse units (I MU = 5.2 j.lgextract). Error bars indicate one stan­dard error of the mean (n = 8). Curvedefined by the dotted line representsthe administration of fish ciguatoxinas reported by Tachibana (1980).

dose dependent. A nonlinear equationestimated the relationship between theindependent variable (dose) and the de­pendent variable (time-to-death). Usingthe Simplex method for determining thevalues of the parameters by leastsquares, the following equation gave areasonable approximation of the curvein Figure 2:

Dose (MU) = 0.8 (time-to-deathin hours)-l.

Coupled with differences in sensitivityto orally administered toxins, themarked difference in time-to-death re­sponse to ciguatoxin reported by Tachi­bana (1980) and that obtained in ourstudy with G. toxicus extracts further il­lustrates that the two toxins may not beequivalent biologically. The dashed linein Figure 2, superimposed on the dino­flagellate response curve, represents acurve based on the formula reported byTachibana (1980) for ciguatoxin:

Log dose (MU) = 2 log (1 + time-to­death in hours)-l.

The differences between the fish-toxin­derived curve and the dinoflagellate­derived-curve are evident from thisfigure.

Estimation ofToxin Retention Time

Our knowledge regarding the ability

48(4), 1986

80

~ 60c:o0.

'""~ 40C'""Q;

Cl. 20

• •0.5 1 2 4 8 16 32

Interval between injections (hours)

Figure 3.-Percent response (death)within 48 hours following three equal­ly spaced i.p. injections of G. toxicusextract.

of the animal to clear, sequester, orotherwise inactivate ciguatera-associ­ated toxins is vitally important to ourunderstanding of the human illness,ciguatera. Unfortunately, direct methodsof determining the biological fate ofthese toxins is not readily available. Togain insight as to the "half-life" of thedinoflagellate toxin, portions of anotherwise lethal dose were administeredover several time intervals in order toextrapolate the time point in which theeffect of individual doses were no longeradditive (Fig. 3). A control group whichreceived a single 10 I-Ig standard dose ofthe toxin extract elicited the expected 75percent lethality response. Another con­trol group received a single 3.3 I-Ig doseand exhibited no fatalities. Groups thatreceived three injections of 3.3 I-Ig eachat time intervals ranging from 0 to 4hours between injection gave between40 and 60 percent response, while thoseanimals in groups injected at intervalsbetween 8 and 30 hours gave less than10 percent response. From these results,it appeared that an estimation of bio­logical half-life for this toxin in themouse model lies somewhere between4 and 8 hours, and that this toxin maynot accumulate in the body.

The persistence for months of theneurological symptoms associated withciguatera poisoning in humans certain­ly suggests that either ciguatoxin is re­tained and remains active for longperiods of time or that the damagecaused by ciguatoxin is not quicklyrepaired. As yet, a similar "half-life"study using crude extracts of ciguatera

fish has not been performed. It will beof interest to know if ciguatoxin, unlikethe dinoflagellate toxin, is retained in themouse model.

Biological studies of the toxins asso­ciated with ciguatera have been mini­mal. However, many of the biologicalproperties described can be very usefulin defining the various toxins that havebeen isolated. As a clear understandingof the biological activities of theseciguatera-associated toxins is obtained,their relationship to each other and amore rational approach to minimizingthe impact of ciguatera may becomeevident.

Acknowledgments

We thank Marilyn Orvin for her tech­nical assistance, Rick York for supply­ing extracts of the dinoflagellate, and H.Hugh Fudenberg for valuable discus­sions. This research was supported inpart by NOAA Grants NA80AA-D­00101 and NA84A-H-SK098.

Literature Cited

Bagnis, R., T. Kuberski, and S. Laugier. 1979.Clinical observations on 3,009 cases of cigua­tera (fish poisoning) in the South Pacific. Am.1. Trop Med. Hyg. 28:1067-1073.

Banner, A. H., P. 1. Scheuer, S. Sasaki, P. Hel­frich, and C. B. Alender. 1960. Observationson ciguatera-type toxin in fish. Ann. N.Y. Acad.Sci. 90:770-7lr7.

Hoffman, P. A., H. R. Granade, and 1. P. McMil­lan. 1983. The mouse ciguatoxin bioassay: Adose-response curve and symptamology ana­lysis. Toxicon 21:363-369.

Sawyer, P. R., D. 1. JoUow, P. 1. Scheuer, R. York,1. P. McMillan, N. W. Withers, H. H. Fuden­berg, and T. B. Higerd. 1984. The effect ofciguatera-associated toxins on body temperaturein mice. In E. P. Ragelis (editor), Seafood tox­ins, p. 321-329. Am. Chern. Soc. Symp. Ser.262, Wash., D.C.

Schantz, E. 1., 1. D. Mold, D. W. Stanger, 1.Shavel, F. 1. Riel, 1. P. Bowden, I. M. Lynch,R.1. Wyler, B. Reigel, and H. Sommer. 1957.Paralytic shellfish poisoning. VI. A procedurefor the isolation and purification of the poisonfrom toxic clam and mussel tissues. 1. Am.Chern. Soc. 79:5230-5236.

Scheuer, P. 1., W. Takahashi, 1. Tsutsumi, and T.Yoshida. 1967. Ciguatoxin: Isolation and chem­ical nature. Science 155:1267-1268.

Tachibana, K. 1980. Structural studies on marinetoxins. Ph.D. Thesis, Univ. Hawaii, Honolulu,157 p.

Yasumoto, T., I. Nakajima, R. Bagnis, and R.Adachi. 1977. Finding of a dinoflagellate as alikely culprit of ciguatera. Bull. Jpn. Soc. Sci.Fish. 43:1021-1026.

---=_-,--' I. Nakajima, Y. Oshima, and R.Bagnis. 1979. A new toxic dinoflagellate foundin association with ciguatera. In L. Taylor andH. H. Seliger (editors), Toxic dinoflagellateblooms, p. 65-70. Elsevier Sci. Publ., N.Y.

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