Response of Mice to Gambierdiscus toxicus...
Transcript of Response of Mice to Gambierdiscus toxicus...
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.
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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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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.
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'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
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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.
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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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