Upload
vanthuy
View
231
Download
6
Embed Size (px)
Citation preview
/q
AD____
MARINE BIOTOXINS: LABORATORY CULTUREAND MOLECULAR STRUCTURE
I% uAnnual Report
Paul J. Scheuer, Ph.D.
October 18, 1989
Supported by
U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick# Frederick. Maryland 21701-5012
Contract- No. DAMD17-87-C-7210D"TIC
University of Hawaii at Manoa ELECTE2545 The Nal•, NOV 1598
Nonolulu, Hawaii 96822 4,
DOD DISTRIBUTION STATEMENT
Approved foe public releasel distribution unlimited
The findings in this report are not to be construed asan official Department of the Army ponition unless sodesignated by other authorized documents.
8'9"1.1 14 001. .•
$11CURElN CLSStFI TION14 OF THIS Mr
REPORT DOCUMENTATION PAGElI. PORT SECURITY CLASSIFICATION lb. RESTRICTIVE MARKINGS
Unclassified _
IS. SECURfTY CLASSIFICATION AUTHORITY 3. $ISTRIuTION/AVALA&ILITY OF REPORT
2b. DECLASSIFICATIONIDOWNGRAOING SCHEDULE Approved for public release;distribution unlimited
4. PERFORMING ORGANIZATION REPORT NUMBER(S) 5. MONITORING ORGANIZATION REPORT NUMBER(S)
to. NAME OF PERFORMING ORGANIZATION 6b. OFFKE SYMBOL 7I. NA•IE OF MONITORING ORGANIZATION
University of Hawaii at Manoa Ofk )
OREDIS (C0ty, Stft., and ZAP C**) 7b. ADDRESS (City. Stote, and ZIP rode)
2545 The MallHonolulu, HI 96822
Be. NAME OF FUNONG/SPVNSO.tING 6b. OfFICe SYMBOl. 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER
ORGAMZATIONU.S. Army Medical (WV 8 abbu)Risearch & Development Command Contract Wo. DA)1),7-87-C-7210
&L ADDRESS(Z;, Stat end III Coob) IA SOUrts Of FUNIING NUMBERS
PIDGPA@M XCT TASK IWORK UAIT-Fort, Detrick .LIEMINT NO. V No. IACCESION NO,Frederick, MD 21701-5012 62770k 162770A871 AA 399
1I. TITLE 0d Sftw"t 0800GOON)
Marine Biotoxins: Laboratory Culture and Molecular Structure
12. PRRSONAL AUTHOR(S)
ScheuerI Paul JosefA )..TPO REP 136 .II COvEEt 14. DAEa;01 Nw. IPAGE ONIAnnual IROM9158TOV1L89 1989 October 18 ...... 371t. SUPMMIENTARY NOTATION
17 COSAfl CODES I SUBJECT TIPM (CaPPUve an rewueId IcgI y A*fy by iiick =u.awFIELD GROUP SUB-GROUP Ciguatoxin,' Maitotoxin,' Pelytoxin; Cambierdiscus Toxicur,
06 13 . MamJn Toxin,' Lab Animals,' RA I f -06 03
14, ABTRA lCont*'we on, rqW" of neenvy =n MwIV yboch nMbf)
" Toxic cultures of Cambierdiscus toxseus have been established. Stoassays (nouselethality) and pharmacological profiles indicate that the toxins are maitotoxin anda toxin vhich has the pharniacological profile of cigiatoxia but has reducedlethality.These results must be confirmed by replication. Small amount* of ciguatoxin are beingpurified from mooray eel viscere procured on Tarava atoll, Kiribati. and from locallycaught yellow amberjack, Seriola dumorilil . .
20. ODSTkIUTIONIAVAiLAWILITY Of ABSTRACT 21 ABSTRACT S•LURITY CLASS4fATAION
DLNOCLASSIFIEO&JNLIMITED0 C SAME AS RPT 0 TIC USERS Unclassified110 NAW RCSPON$4,BE. INDIVIDUAL 22b TELEPHON1(bw*• eCoJI -2c OhFIC $'MB
Mrs. Virglnia H. Miller. 301/663-7325 SGRD-R)I-S
00 Form 1473. JUN 6 p'ow or* ev0e oso0•.1. SECURITY CLASSIFICATION Of NI PA
p o
FOREWORD
Opinions, interpretations, conclusions and recomendations are those of theauthor and are not necessarily endorsed by the US Army.
Wt.ere copyrighted material is quoted, permission has been obtained touse such material.
Where material from documents designated for limited distribution isquoted, permission has been obtained to use the material.
Citations of commercial organizations and trade names in this report3o-no"t constitute an official Department of Army endorsement or approval ofthe products or services of these organizations.
In conducting research using animals, the investigator(s) adhered toi'WGuide for 'the Care and Use of Laboratory Animals,* prepared by the
Committee on Care and Use of Laboratory Animals of the Institute ofLaboratory Resources, National Research Council (NIH Publication No. 86-23,Revised 1985).
For the protection of human' subjects, the investigator(s) adhered tooTTcies of applicable Federal Law 45 CFR 46.
1n conducting research utilizing recombinant DNA technology, theTnvestigator(s) adhered to current guidelines promulgated by the NationalInstitutes of Health.
PI- STnatur*'
A-eset fn leer
MeTI TABUnumoeuced
Justritlbcation.------./
Avollb stT od/orDit Seial
TABLE OF COfNTENTS
I. Introduction 2
A. The Problem 2
B. Current Status 3
C. References 4
II. Results During Year II 5
A. Dinoflagellate Culture .5
B. Ciguatoxin Isolation 17
III. Conclusions 19
IV. Appendices 20
* -2-
I. UITROWCFION
A. The Problem
Marine biotoxins are among the most potent naturally occurring toxins
known. Their physiological actions are diverse, as are their molecular
structures, not all of which hay been fully determined. Some of these toxins
constitute a hazard to human hea th and safety. It is therefore imperative to
elucidate their structures and t make pure toxins available for the study of
pharmacological properties and hanisms of action.
In this study we are prinripally concerned with two toxins that are
associated with the human fish intoxication known as ciguatera, ciguatoxin and
umitotoxin. and with palytoxin. Each of the three toxins presents a different
set of problems.
1. Ciguatoxln (CTX)'
Its molecular structure of ctguatoxin (1) has recently been determined by
Yasumoto and coworkers (Personal Coimmnlcation). The compound is a 55-carbon
chain fashioned into 13 contlguous ether rings, reminiscent of okadaic acid
and the brevetoxins.
M..F
1It 3 OH (Ibortay eel)
.2 f' c ,,C.c- (G. ,.o5icuo)
n 2
Laboratory culture and continued extraction of eel viscera are necessary in
order to
a. study the poorly known pharmcology and chemistry of the toxin
h. devise a monoclonal antibody-based diagnostic test to detect
ciguatoxic fish
c. determine the absolute stereochemistry of the compound
d.. initiate molecular modelling studies directed toward drug development
and therapy.
2. K.i totoxin
In contrast to ciguatoxin, which is soluble in organic solvents.
aaitotoxin is water-soluble. It was first described from the gut of
herbivorous toxic fishes, where it occurs in low concentration. It has been
produced in a number of laboratories from cultured . toxicus. Its molecular
structure is partially known.
3. Palytoxin
This toxin, whose structure has been known for several years and which
has, recently been synthesized', has so far been isolated from toxic zoanthid
corals, Paltho sp. Preliminary evidence suggests tfhat the producer of this
toxin is not the coral, but an epiphyte. possibly a bacterium.
B. Current Status
1. Ciguatoxin
The PI reviewed the status of ciguatera research at the Mycotoxins and
Phycotoxins Symposium "n August, 1988 in Tokyo, Japan. A copy of the paper is
attached as Appendix II. The principal development since then is the
1
-4-
structural elucidation of ciguatoxin by Yasumoto and coworkers (Personal
Communication). As a result, ciguatoxin production can now be devoted to the
tasks outlined under Section A.1 above. For work in Hawaii the best toxin
source remains eel viscera from Tarawa atoll. Republic of Yiribati. despite
formidable communication and transportation problems.
There are preliminary indications (see Section II a. Table I and Figure
2) that our laboratory culture of t. toxicus produces a toxin that has a
pharmacological profile of ciguatoxin but is less toxic than CIX. If these
experiments can be replicated, production of this toxin will be arz important
goal.
.2. Maitotoxin
Replication of experiments tc evaluat:e our C. toxlcus cultures (see
Section IIA), followed by quantification and upscaling will allow us to begin
maitotoxin production in the near future.
3. Palytoxin
We have not yet initiated research on this phase of the contract. We
will recruit appropriate personnel within the next few months.
C. References
1. Kishi,-Y. et al.. .1. Am. Chem. Soc. 1989, Ill. 7525, 1530.
II. Results During Year II
A. DINOFLAGELATE CULTURE
1. INTRODUCTION
Previous reports have detailed the attempt. to establish cultures of the
benthic dinoflagellate 0amblerdiscus toxicus and associated dinoflagellates
under laboratory conditions. The results of these attempts are now presented,
with emphasis on the progress achieved since the lait Quarterly Report was
submitted.
2. MATEMIALS AND MHOEDS
2.1 Population densities of benthic d noflJ.ellates
The methodology used to determine the population densities of
dinoflagellates growing attached to macroalgae is as described previously.
Briefly, species of macrolgae were harvested in approxisately 50g quantities
from Kahala Beach. Oahu. The algal samples were shlken in seawater and the
blodetrital material so obtained was filtered successively through nylon
filters of mesh size 250. 100 and 37 pro. The number of calls of mkblerdisru
toxlcus and other dinoflagellates in I ml, aliquots of the biodetritus fraction
(100 - 37 jzm) was determined using a calibrated water counting chamber.
2.2 Culture of dinof1aiellates
Batch and clonal cultures of Gambierdiscus toxicus and associated
dinoflagellates have been initiated as follows. Cells of dinoflagellate
1
-6-
species are removed frtm the algal biodetritus with sterile micropipettes and
are washed in sterile seawater. For clonal cultures, single cells are
transferred to sterile test tubes containing 5 mL of ES medium (Provasoli.
1968). For batch cultures 20 cells are placed in 10 mL of ES medium. The
cultures are maintained at 27"C on a 14:10 light:dark cycle at 1.OxlO's quanta
ca"2 s81 provided by "Cool White" fluorescent tubes (General Electric). At
three weeks, the cells are transferred to 125 mL flasks containing 50 ml. of ES
medium. Culturos so established are maintainod as stock cultures in ES medium
and subcultured every 3 - 4 weeks.
Axenic cultures of each clonal and batch stock culture are obtained by
the following method. Samples of each stock culture (100 - 200 cells) are
washed in sterile medium using glass spotting dishes and transferred to ES
medium supplemented with antibiotics (Penicillin:Streptomycin; 2:1). After 48
h. the cells are placed in a 50 mL flask containing 20 mL of normal ES medium.
This process is being repeated at 3 - 4 week intervals.
2.3 Harvesting and extraction of dinoflaxellates
For toxicity studies, batch and clonal cultures of Gambterdiscus toxIcus
were prepared as follows. Fernbach flasks each containing 1.5 L of ES medium
were inoculated with seed cultures of the dinoflagellate (approx. 200 mL; 200
cells/mL). The cells were maintained for 3 - 4 weeks as decribed previously.
at which time the cell density had reached 1 - 1.5x106 cells/mL.
The cells were harvested by filtration onto glass fibre filters (Whatman
CF/A). washed with deionized distilled water to remove salts aid lyophilized.
77
The washed, dried cells were sonicated in acetone and extracted in acetone
over 48 h. The extract suspension was filtered and the acetone-insoluble
material extracted in methanol over 48 h. Solvent was removed from both the
acetone and methanol extracts under vacuum (Buchi. Rotavapor) and the residues
taken to dryness under a stream of nitrogen. The crude extracts are being
stored at -20"C.
2.4 Mouse biossa
Male and female mice (Swiss white: 18-23g) were injected i.p. with doses
of the dinoflagellate extracts prepared as fine suspensions in 1% Tween 90 in
physiological saline. Signs expressed in the mice following administration of
the extracts were identified according to the criteria detailed in Hoffman
et &l. (1963) and recorded at I h and then at intervals up to 48 h. The mice
were used and sacrificed in accordance with ethical standards.
2.5 Cardiotoxic effects of extracts of qambierdiscus toxIcus
The cardiotoxic effects of selected extracts prepared from cultured Q.
toxicus have been evaluated usixtg the following method. Male guinea-pigs
(300-500g) were sacrificed, the hearts excised, and placed in oxygenated
Krebs-bicarbonate solution. The left and right atria were dis'sected free and
used separately to study the effects of the t. xcus extracts upon the
electrically-stimulated and spontaneously-elici ted contraction, respec.tively.
The left atrium was stimulated by rectangular pulses (1.5 threshold voltage. 4
msec duration and at 1.5 Hz) delivered through a Grass SD9 stimulator. The
isometric responses of both the left and right atria were measured using force
transducers and recorded on a Crass polygraph. The crude extracts were
dissolved in 1O0 methanol and 100 pzL aliquots of the solutions agded to the
organ bath to give a final concentration of 10 Wg/mL.
3. IJSULTS
3.1 Potulation densities of benthic dinoflatellates
The population density of Gai•iterdiscus toxicus and OstreoQsts
lenticularis growing attached to macroalgae collected at Kahala Beach. Oahu.
has been monitored from September. 1988 to August. 1989. The results of this
survey are presented in Fig. 1. There has been a gradual increase n the
number of both g. toxc-us and Q. lanticularis growing attached to the r d alga
Spridia fLil ntosM since March, 1969. with the maximum number #,% cells of
each species of dinoflagellate being reco;'ded in August. 1989. Analysis of
biodetrital fractions obtained in June man Aurist. !W9 revealed the presence
of both .g. to&icus and Q. jetjjljj growlog attadhed to Anthophora
sicLt•ra and Dictyota sp. On one previous occasim.o. Q. J .ta;jarjj had been
found in the bitdetritus obtained :rcmA s.iciee . wherea6 G. h-j.u_ had
been found growing only on a. L•1in.tosa. Other workers (&wbor %.• IL..
1969); Gillespie et Md.. 1985) have reported that IL.jLc ,isp'eys an
apparent preference for certain species of Nacroalgae. particvlarly those that.
possess a filamentous and/or branched morphzlogy and therefore a large surface
area. However. it has also been demonstrated that C,. toxicus will utilize a
wide variety of algal substrates, including species from different divisions
(Bomber _e al.. loc. OA., Gillespie e.jtj., jo. cit.).
1. , JQS. Q
2000
oUtii1000O
"Iu
SONDJ FMAMJ J ASOMONTH
Fig. I The mean density of Gambierdiscus toxicus [I] and Ostreousislenticularis (i ] growing attached to the alga Spyridia filamentosa. Countswere made of the dinoflagellates present in biodetrital material (37 - 100 Pinfraction) obtained froma plants to S. filamentosa collected from "Kzhala Beach.Oahu. The mean densities were calculated from cell countE obtained from fiveplants of S. filamentosa. Standard error bars are not shown.
- ic - /'
3.2 Dinoflazellate culture
An inventory of the dinoflagellate cultures currently being maintained in
the •hemistry Department is presented in Appendix I. Six species of benthic
dinoflagellate are being cultured, with the'emphasis on the culture of both
batch and clonal cultures of Cmbierdt scus toxicus and QOrtrosA
lIntlcularis.' Cultures of Q. toxicus have been initiated from biodetrital
samples collected from Hawaiian and Tahitian waters. A total of eight batch
(non-clonal) and 34 clonal cultures of Q. toxicus and one'batch and 28 clonal
cultures of Q. int1cularis are currently being maintalned in ES medit".
Certain cultures have been found to acclis te better to laboratory conditions
than others. Cultures of . ttsoxi that are growing well under the
conditions descrilbed in the Materials and Methods section include clorml,
cultures CT 41. 158. 174. 177. 181. 196 and 188 and TCT Stn4/2. Division
rates of 0.42 divisions/day have been rece.-Aed for clone CT 188. while for the
other cultures the division rates range from 0.23 divisions/day to 0.38
divisions/day. Frequent subculture of'all cultures is undertaken so as to
achieve stable growth under laboratory conditions. The other four species of
benthic dlnoflagellate beitn grown are PrarinentrUS JUI. f. qn£MTa.
Amohidinium sp. and M gonotts. Partially axenized cultures of all batch
and. clonal cultures have been obtained. Whilst there is no evidence of
large-scale Injurious bacterial contamination of the stock cultures, efforts
will continue to obtain totally axenic Cultures.
3.3 Toxicity ofetract,_ of cultured d1rUt
Extracts obtained from both batch and cloral cultures of CqjmhJrdJLi
j.•,. end cultures of Q2 12C 9 jr JnJu.rJA and r JIM. have
*. " -II1-
been evaluated for toxicity using the mouse bioassay. Data pertaining to the
cell and crude extract yields for one liter cultures of these dinoflagellates
are detailed in Table I. The weight of lyophilized cells obtai.ned has been
found to vary both for liter cultures derived from the same stock culture and
for those grown from different stock cultures. These discrepancies my
reflect the degree of bacterial xmotamination %asociated with individual stock
cultures. For example. cultures that are only partially axeniezd tend to
produce large amount of mucous and the we'fgt of cells obtained is
considerably higher than that recorded for cultv we such as CT 41 and r. La..
KT 16/1 that are testing bacteria-free. Both the mttamnol and acetone
extracts of cultured G. toxics have proved to be toxic when tested in the
mouse bioassay. In the case of the acetone extracts, the only sign of
toxicity evident in the mice have been the onset of severe diarrhea, with all
of the mice surviving the 46 h test period. The methanol extracts have proied
to be far more toxic. inducing a range of toxic signs in the test animals
including ataxia. wobbling gait. reduced forelimb grip, lose of body tone with
associated hypothermia, cyanosis and breathing difficulties. Death
accosminied by terminal convulsions has been recorded at 4 h
post-administration of the extracts. with the primary cause of death being
respiratory paralysis. Methanol extracts prepared from culture- of
rroct•t.Q..rm ýUn have also proved to be lethal to mice at a dose (i.p.) of 10
qm/kg. E:.tracts of one culture of QIIRWgp lent icjljds were not toxic to
mice.
-12-
Table I. Cell and Extract Yields, Toxicity to Nice of Crude Extracts fromzi tural Dinof lagel late*'
CULTURE YIELD OF YIELD OF EXFRACr (mg) TOXICITY TO NICEcawS(w•) A R A N
(A) 23.4 13.0 24.0 * o(L)(B). 35.6 1.6 5,0 * o(L)(C) 72.8
. l. ;2B 2/28 110.2
G..s. ;KB 64nh 75.5
Tg. t.; Stn 4 22.2 13.0 13.4 0 0(L)
Q. I=.; Xi 1/18 67.5 11.4 21.8 - -
TCT Stn4/2 232.1 17.0 14.2CT 41 72.0CT 174 242.2 22.0 14.0CT 177 219.9 '12.0 16.0 0 0(L)
PL 4 (A) 90.3 41.3 7.9, - (L)P1. 4 (B) 106.3 12.1 6.4 - 0(L)
rThe yield of cells denotes the weight (mg) of washed lyophilized cellsobtained per liter of dinoflagellate culture. The symbols A. B and C refer toseparate liter cultures of the same stock culture of a particulardinoflagellate species. The coding system used for the dinoflagellates is aslisted in the inventory of dinoflagellate cultures (Appendix 1). The yield ofboth the acetone (A) and methanol (N) extracts is exprested in v. Thetoxicity of each extract was evaluated using the mouse bioassay. The dose ofthe crude extract adtinistered 4.p. to the mice was 10 mlA/k. The symbol 0I
indicates that the extract induced toxic signs in the test animal. while thesymbol L indicates that the extract proved to be lethal.
"-13-
3.4 Cardiotoxic oroverties of G. toxicus extracts
Both the acetore and methanol extracts of Hawaiian Gambie Ma toxicus
(Batch G. =.. KB 18/1) have been found to be cardiotoxic when tested upon
the isolated guinea-pig atrium. Representative traces obtained in the course
of these experiments are presented in Fig. 2. The acetone extract (A.
10 pg/mL) of r. toxicus exerted a positive inotropic effect upon both the
electrically-stimulated (Fig. 2; panel 1) and spontaneously-beating atria
(Fig. 2. panel 3). This extract also elicited a positive chronotropic effect
upon the spontaneously beating atrium (Fig. 2; panel 3). The positive
inotropic effects of the acetone extracts of t. lxim were blocked in the
presence of the adrenergic blockers propanalol and phentolamine and the sodium
ion channel blocking agent tetrodotoxin. The effects of the g. .icus
acetone extract upon the isolated guinea-pig atria closely resemble those
described for ciguatoxin and for extracts of certain species of toxic fish
(Xiyahara 2t &L., 19W). The effects of the methanol extracts (N; 10 pg/mL)
of G. taxicus upon the electrically-stimulated guinea-pig artrium are shown in
Fig. 2. panel 2. The extract induces a positive inotropic response similar to
that recorded previously for mitotoxin (P, of. Y. Hokasa. pers. comm.).
4. COV3USIONS AND OMMING qUDIES
In previous reports, it was noted that problems encountered in providing
a suitable environment for the culture of benthic dinoflagellates. was
hampering attempts to establish cultures of these microalgae in the
laboratory. These problems have now been largely overcome and cultures of •.
txicus and associated dinoflagellates are being successfu!ly ,.Nintained in
-14 -
4 W3
449 "6
4~C4
44.0 - 0~
J0
#4 .. es
-15-
the laboratory. The primary objective of the ongoing work is to optimize the
growth of the se cultures and to screen as many as possible for the presence of
toxic substances. Those cultures that have already proved to be toxic in the
souse bioas y are being cultured in larger volumes so as to provide
sufficient qt antittes of the crude extract for the purification of the toxic
components tc be attempted. Results obtained using the isolated guinea-pig
atrium suggest that a ciguatoxin-like compound(s) my be present in extracts
of Hawaiian 'j. toxtcus. It is also likely that cultures of Q. toxicus are
producing matotoxtn as has been reported for cultures of Q. toxicus grown
elsewhere. F'urther experimentation is needed to ensure that this is indeed
the case. It is also planned to continue to study the optimization of the
growth of cultures that prove to be toxic in the bioassays. Experiments
dealing with modifications to the culture media supporting the growth of the
dinoflagellates have beer. initiated. Another series of experiments designed
to evaluate :he use of different substrates to facilitate the growth of r.
toxicus are planned. Of particular interest here will be the use of
microcarriers: these are small beads composed of a variety of materials
including gls~s and plastic, that have been used in cell culture to facilitate
the growth oO anchorage-dependent cell lines. If these microcarriers can be
used in a similar fashion for the growth of G. toxicus, this would greatly
improve cell yields and thereby increase the supply of extract required for
the purification of the toxic components.
-16-
5.- LITERATURE CITED
BOMBER. J.W.. RUBIO. G.G. and NORRIS. D.R. (1989) Epiphytism of
dinoflagellates associated with the disease ciguatera: substrate specificity
and nutrition. P, (in press).
GIL•ESPIE, N.C.. HOLMES. N.J., BURKE. J.B. and DOLEY. J. (1985) Distribution
and periodicity of Cambierdiscus toxicu$ In Queensland. Australia. In: 1oxic
DInoflaxellates (Ed by D.N. Anderson, A.W. White and D.G. Baden) pp 183 - 186.
Elsevier Science Publishing Co., Amsterdam.
HOFFMAN, P.A., GRANADE, H.R., and NcMILLAN, J.P. (1983) The mouse ciguatoxin
bioassay: A dose - response curve and symptomatology analysis. Toxico 21,
363 - 369.
MIYAHARA, J.T.. KAMBAYASHI, C.K. and HOKAMA. Y. (1989) Pharuacological
characterization of the toxins in ciguateric fishes. In: Mvcotoxn n
Phycotoxtn-3 8 (Ed by S. Natori, K. Hashimoto and Y. Ueno) pp 399-406.
Elsevier Science Publishing Co., Amsterdam.
PROVASOLI, L. (1968) Media and prospects for the cultivation of marine algae.
In: Cultures and Collections of algae. JM. So&. El. Physiol. pp. 63- 75.
-17-
B. Ciguatoxin Isolation
Approximately 500 kg of Yellowtail Amberjack. Seriola dumerilii. was
extracted whole to shorten the extraction process and maximize total
ciguatoxin yield. A total of 70 pg of crude ciguatoxin was obtained as
determined by toxic reactions in mice. However, further purification and
testing indicated that substances in the crude isolate other than ciguatoxin
W contributed to the toxicity. Thus whole fish extraction yielded roughly
the same net amount of ciguatoxin as extraction of the fish viscera only.
Since the purification process had to be lengthened, whole fish extraction
became more time-consuming than visceral extraction. It was then decided that
subsequent extractions would be performed on the viscera only.
Subsequent visceral extractions of S. dumerilli also yielded small
amounts of toxin and required many purification steps. Thus in June, 1989, it
was concluded that only small quantities of toxin could be isolated from S.
d merilli, and only at great effort.
In June. 1989. we resumed procurement of eel viscera from Tarawa despite
continuing communication and transportation problems. A shipment arriving on
July 5, 1989. contained 1.65 kg of moray eel viscera, from which 8.5 Pg of
crude ciguatoxin was isolated. The concentration of ciguatoxin isolated per
kilogram is roughly equivalent to that derived several years ago. Thus
cipgatoxin' levels in eels from Tarawa has remained constant, and Tarawa
remains an excellent source of ciguatoxin-containing eels.
Locally, recent ciguatera outbreaks had been reported, which prompted a
collection of moray eels from the Hyatt Regency Hotel beach in Kona and Anae
Hoomalu Bay. Haweti. These eels were received via Dr. Hokama's laboratory and
S.'" ' . - 18-
were extracted for ciguatoxin. The mouse assay tests of the crude extracts
resulted in ciguatera-like symptoms in the mice, indicating the presence of
the ciAguatoxin. but in small concentrations. These findings confirm a
possible ciguatera outbreak on the Big Island of Hawaii and also indicate
further exploration a source of ciguatoxin.
Recently the question as to the stability of the ciguatoxin over time was
addressed. In September. 1989, extraction was made of eels from Tarawa that
had remained unrefrigerated in transit for more than one week. The toxicity
of this extract proved to be the same as the toxicity of extracts from freshly
obtained eels. This demonstrated that ciguatoxin remains stable In
unrefrigerated eels for at least one week.
Table II. Ciguatoxin Isolated from Seriola dumerilii and
Moray eels from the Period October. 1988, to September. 1989.
Extract Date wt. (kg) toxin(Ag)
fish flesh 10/88 170 ~12and viscera to 9/89
eel viscera 7/11/89 1.65 8.5(Tarawa)
eel viscera 8/30/89 0.96 -1.5(Big Island)
eel viscera 10/1/89 5.10 54(Tarawa)
A total of approximately 160 pg of toxin previously and currently accumulated
has been prepared for HPLC purification.
." - 19-
III. Conclusions
Gamblerdiscus toxicus culture is now well established. Replication of
recent toxicity assays ard scaling up of toxin production are our immediate
tasks.
Accumulation of ciguatoxin from mooray eels will continue to provide
material for the determination of the stereochemistry of the molecule: for
monoclonal antibody production; and for pharmacology, including drug design.
Palytoxin research will be initiated.
*APPENDIX 1
DINOFLAGE.LLATE CULTURES, CHEMISTRY DEPARTMENT, UNIVERSITY OF HAWAII
SEPTEMBER, 1989
1. s1u l s 3.I Smldia filamtssat 1/11/39 1. Iewtrm.L kilo lahala I. inridt 1hatuat 3/31/31 1. IeAffrovf. taum 1abIe I. hmridtila Z oiwala 4/2/61 1. koCaffrnvI,,Ulm lahalal. $Midifl ms~ton 6/37/31 3. cotgiatLhzlna lakah 1. Di It. 6/7/319 .LCorgtuI. !aim las) . ctopiho nicifera 6/07/l' 3. Corsist
I. hamU ltitlaiota 1al 1 /63 . kwafmrs
cmA CISTI33S
ot 41 l.ah 3. 5hizz1 jfjjggto 1/13/31 I. kWafrn
a 155 lab.). 1. buidl filmiMto 4/r7/311 . kwsffrna15 inb.!. 3. Sonjjj allmhlfu 4MI7/3 J. kWalfreof 157 Laba I. hVlAb filana~m 4/17/19 1. hCairtias?1in labsial. ' mlidaina l f6101 /17/11 1.Corgiat611641 Lab" 3. Jwri4j fl1lmatm 6/17./3 1. Corlist67 173 lab..!. I. Urm dtflowtan)l /6/.7/11 3. Cortlatff 177 lkab. 3. sorj4di ujjnu j m/67/1 1. cortlat11 177 [WWuI& . fmid iliniasag 6/17/31 1. Corsiata7173Ilab.!.1. Smz df fla mo IjjAT /17/1 3. CorgiatIT 131 hi). 1. !nrldk IUAana ~ 1/07/11 3. CoreiatIT186 hab.).3. flmadlaL laluno 1/17/31 1. Cortista71II lakb.) 3. .6rnidi filaunst 1/37/31 1. Corgiata73 lab14Ial. Smidip filmfaoo 6/17/31 1. Corgiat67 113 Ilia"a 3. Srmidisfla ntojj A~ 4/17/31 1. CorgiatIT 211 labma 3. Smdi fijj~o 1/07/41 1. CortiatV7 In IMIS) 3. SLUMj$j fillj)a 1/1719 3. CortiatC 237 Wala a 3. Sjg). film)tos 1/11/19 1. CortiatC7 2I" bda), 3. 5"ridia filasto C/17/11 P. CorgiatIT 215 lawa& 3. Smlidi 111 matou 6/17/81 1. WcOaNTeIT 232 wlabs 3. Iffridia filauestosa 6/U7/89 L1 Ic~ffrysRT 221 habala 3. ;jmjgj, flivettas 6/17,/1l D. Cottiat 4"6 234 habala 3. krzidi filamestoma 6/07/3f1 . CorgistG'? 231 labaia 3. jSzzLdi fljistatol 1/17/89 0. Corliatcy 244 bLa) 3. .JM=) filiunso 6/07/89 D. Cortiat
Turn4400' EmAp
lots hm op. 5/30/89 1. cCafrey
Iota ajj op. 5/3 0/1S 1. IcCaffrtyIT? 76 11HUI,
lots JUU~ op. 5/36/11 I. af cCLrayIC 73 11th',
ot, hIaU It. 5/36/33 1. Kec~Affm
sets LW p. 5/31/IS I. Iewattry
"mto Iaa op. 5/30/13 1. Wafrty
Ost~ephsl leutleularic
.1.Intialainu Idols1a. Sjyridij fillemetoea 1/13/33 1. kWafme
OL 51 W Rhia . Sqyridha filameltou 12/17/13 1. IWfirey0L1212 lkula 3. Acuathopbora aicif era 1/17/11 P. Corliat01 121 Waall 1. Icaatboyborm audicera 6/07/83 3. Corgiat01 123 Waall 1. Icatkopbora spicifers 6/07/33 1. WSWiaOL 12I Walli 1. Acauthophora apicifera 3/37/33 1. Corgiat01 136 Ilalla I. Dictiot op. 6/17/33 3 . Corgiat01 131 Waull 3. Dictyota ap. 6/67/13 3. corgiat01 132 luau 3. Dictrota at. 6/67/SI 3 . corgiat01 134 lahala 3. Dictiota up. 6/37/33 1 .Corgiat01 135 Waall 3. Dictiota op. 6/07/33 3. ~CouldOS 133 Idlah 3. Dictyot qp. 6/07/33 1. Corgiat01 140 hiaula 3. Snyridha filauestosa 6/07/83 3. CorgiatUt. 142 hukla 3. Spyrldi tilaimetooa 6/07/33 D. Corgiat01 144 labala 3. DUfj4 LA Z.li1aiu.lu 6/07/83 D. corgiat01 146 hlaa 1. j07riAdi 11iinin.oo 6/07/33 3. Corgiat01 147 Walal I3. Wrindl filaseatoma 6/07/33 D. Corgiat01 143 Waall 3. Spynidia tilaseltoma 0/07/83 D. Corgiat01 151 lahala 3. 597r.idi ilj~asentu 6/07/33 D. Corviat01. 152 lakala 1. Spiridi filangtou 6/07/83 3. Corgiat01 153 hakala I3. Spjjis flamen~itu. 6/PI/89 P. Corgiat01 155 Wakll 3. SpyzrJ.id f~~iaepo 6/07/89 D . Corgiat01 156 Waall 3. Spynidia filueetoi 6/07/13 D. Corgiat01 156 1a1ala 3. 5pyr~i1, flUasepton 6/07/89 D. Corgiat01153 lailla 3. Spyridia filaseatosa 6/07/89 P0. Corglat01 160 lhala B3. Spiridl~ LnfhienUo 6/07/83 D. CorgiatOL 162 luahu 3. Srindle filavento 1 6/07/89 D. CorgiatOL 165 14121 3. Syrnidia filateptoso 6/07/83 0. "OrliatOL 173 Wllbh I3. Syrhidi flantatoo 6/07/83 0. corgiat
ce I Kebala 3. Spyindle filu~ntosa 447/3 1. VcCaffreyCl 2 Iahala 3. Spynidia Mimaenosa 4/27/89 1. IcCaffrey
pit iti
sets oah p. 5/1UP c~affroy
nl 4 iti..,wtv uin" op. 5/36/81 1. L-Caffrer
(htck) IuOWA 1. Acaatho~hore vilcifurt '3/31/11 1. IceAffrey
W. I iltis,mets bdai op. 1'/3 . lciatfrnv
IV. 2 1tims,"mto oa~ p. 5/33 1 c froy
A22..4IX 2&. Natosa. X Hashimioto and Y. Usno (Eds). gwotoxin and Phw'votozina 88 2A Collection of Invited Papers Presented at the Seventh I nternational I UiAC Sympoesium onMycotuaxna and Pbycowoxins, Tokyn. Japan, 16-19 August 1988.@ 1969 Elaevier Science Publishers B.V., Amsterdam - Pnnted in T'he Nether~ands
C1 AICUA - WHA- WE KYW.W AMl WHAT WE WOULD LIKE TO KNOlW
PAUL J. 93MMUDoeartment of Chemistry, University of Hawaii at Manca. Honolulu. HI gem(USA)
The discovery of Gabeds toXicus in 1977 was the turning p~oint Incipiatera research. Du~ring the ensuing decade great stridei have been smed inour knowledge and understanding of ciguatera. Major advances and remiaininunaolved problems are the highlights Of LIe following discussion.
A recent lead article In Th2 Medical Journal 21 Aujjjjal (ref. 1) is
entitled. "What on earth io ciguatera? " The author %nswers the quemtiwa ty
stating, -Ciguatera Is an Impresslve type of fish poisoning which, mea
experienced. is never forgotten..." In the same journal issue Cillespisfand
coworkers (ref. 2) render a concise account 4of ouar Lt'rrent knowledge and
understanding of ciguatera with emphasis on th., situation in Australia. In
other parts of the world, recent symposia and conferences have generated arn
abuAmnit ýcigumtera lite riture (refs. 3-7). which at times has seemsed to
outpace new findings and insights. This surge .if interest my not necessarily
meanm that ciguatera has become wire prevalent. It may merely he a ref lection
paper I will highlight in roughly chronological order the major advancesonatnec fsinit ob trce opzln rbes andti
the principal unsolved riddles, rather than attc,3pt to present yet another
-comprehensive" review of ciguatera.
HISTORICAL NOTES5I Modern scientific inquiry originated in Japan prior to World War 11 with
* Hiyamas (ref. 8) detailed report on poisonous fishes in the South Pacific.
After the war, following a few early fragmentary studies (e.g. ref. 9) by the
late Professor Haeshimoto of Tokyo University ciguatera resea'ch in Japn
* vigorously resumed in the mid-1960's. an effort that Hashimoto's student.
Professor Yasumoto of Tohoku University. has continued with great distinction.
* World War II, specifically United States Involvement in the Pacific.
sparked Bruce I'alstead's interest In ciguatera and led to his landmarkc three
volume treatise published between 1965 and 1970 (ref. 10). It remins the
definitive source of the history of ciguatera. The large list of potentially
cigeatoric fiahes (amt then 400). frequently quoted. perhaps ref lects theglobal distribution of ciguatera. but manzy of the cited reports have net beenrigorously msf treed br modern techniiques. Hiyans' estimates (ref. S) offewer then a hundtred poisonous speces. to probably such closer to the mark.S1imilarly the late Professor eanner a military service in the Pacific &I**roused hit itsterest is cigtmtore. No initiated a reasearch program at theUniversity of Hawai In the aid-lM60a. Although trained io descriptive
mar ine biology Professor Banner reicognized the multi faceted nature of
ciguateirs, and recruited a staff that included ecologists. phamanologist*. "Wchemists. I becm ow of his early recruits.
DIKUDVDY Of G&HILEB DI TCIfLSIntuitive guesses, and empirically supported hypotheses of a diet-deolved
toxin were biked on ebeervat ions of the sporadic eccurreewe of Intoxicationssend of the anatomy mi feedig habits of reef fishes that hod been prowentoxic by 'Incontreworti~let evidence,. it V IN, * CONeumptIOR. swfi'shypothesis (Rot. II) was the (firet comprohoalIve end sell articulated theory
of the origin of cigatotra. It was the result of extensive field studies,both ecological and Schthyfoloslcai. In the Society Islends and in theCaribbea~n. From his abeervatione. f~os interviews with local people. and fromliterature roseearh, ON -1 concluded that three type* ef fish teed to'becom
ciguawter - herbivorous reeof fish,.etts-edn reef fish. meA lorgecarnivores that feed en these two types of roof fish, ontes he postulatedthat the talc orpossis would be bonthic &no "wruwad west likely he en alga, afungus, a prototow, or a bscterlum." Furthe"rmar. %a also would hews to befine. perhaps a blae-greten (cysreoplyte). In ordert to e cOut. for theseemingly rsnAwom outbreaks of ioigatormr in atIetoiusly simoitoic trenat SMandl
also oe~oeted thaet the appearance and growth of the toxic 'erimaelse was the
consequenece of noe or 4e&v%4Q set fie-oo produced by eastural or ea%-eade *ewt.n
Il0-owewr. subsequent field studies designed to btalt noeprimental proof for
thi, new, surfato theory duirng the 11WO) at a ciguatokie reef io Tahit I fail."d
to provide evidence., Noe additimtal field -ir Isieroaery sesdiot hews been put
forth to bolster or refutt the hypothesis~ fete ircusetaaetial evAenc insupoort of a~hnow surfs"the eory cortfinuet to case to light.
Diiieowety of the predicted toxtic precureor. pieoee to be 'eluaive for
rnerly twenty years. Despite Mnxy diligent eforts, which ltheltlid*-,I natioe of stonsch content* of numrous touic fisli*9 (ref 12) and careful
ecological. Ichtyoiogical. and biachwemical scrutiny of herbivierouo sturgeonofishwo (ref, 13) wtetche &I* often the cause of, h~mm ieositeAtion. noe irwec
algae, fungi. or barttenS were fourA T~he decisive breakthrough, by lssuanto
adcomonikera (ref. 14) initintod a row photo in ctiguatora resonrvh
Nicrooople ýirmtiam of a somle of coral detritue from a toxic area in the
Gamier IslnI~ds. Frena Polynesia, re eled that toicity of the samle was
proportional to the nmber 'of cells of a new dineflagellate. DijJassli op..
subsequaently Cambij~jaricus jgjjW (ref. 15). Fractionation of the
detritus Mill purificauion of the dinoflogoiiate toxin to an WD of 56 igAqg
ehowed that two toxin couid be separated. and that they exhibited
* chromatographic behavior that paralleled those of clgiuommsta and initotouin.
Is toerm of Mmse mlte. maittontain predoetiated by a ratio of 3.6:1. Sifts
initeteatim Is approximately three tise- sore toxic then eigpmteman (ref. 16).
the two taulas contributed to toxicity in a nearly *qtl weight basis. At the
time. betb tomins war* but incompletely cha.acterized. my tow the Misr
offersv Wa been directed 'toward etriactueal elucidat ion of sigiitnmeta. the
touin that accumualates in large toxic carnivorouas fish. ibitourxni first
described fro, the etmah centenits of a Tahitian surgeomfish (jWjj) (ref.
17) was lowon and recogizsed by Its water solubility. but bemause of the small
site of the surgeafmtish ma event more difficult to procure' theni eigomteuis.
ftt all wild pepulations of 0. tomiu.as elaborate cipastaxin. ,In
Australia. Cilloottle 21 11. (ref. 19) found that G. twlu from Flinders Reef
(270 latitude) in Southers, (Asoerieland contained high cmncentrations of
wote-soluble but no lipid-soluble toxin. In a IP-outth study they also
demmonstrated that G. uag pspu!at ions psaltool in Seetomber. coinciding with
low water tempratures.
Neoults 'of a Study is Ibeati (ref 19) paralleled thoe" from French
Polynesia (ref. 20). The water-soluble prao~minsted over the other-soluble
touts fraction io a ratio of 26 1. but the overall level of tooicity was
distinctly lower, It is not known whether this is an envirensentai or geneiie
phenmomeno. , Anoher Interesting differenace ~o~ to light lit o t'ae Ill
investigation. In a survey in French Polynesia. Yasunato and cfwerorsr (ref.
it) observed preferential settlinrg of g, toiu an a red AIPi. JgUU~ OP..
while the Hawaii worker* decusanted preferential settling an the red algo
~swr [fLInniUam.wild pop;ulation.i of g jaiu from the Caribbean (ref. 22) alse
furnished lipid-soluble and water-soluble, taste fractions. but the
lipid-soluble material did not elicit symptome In &I"o which at# norwally
associated with thoe" produced by fish-*xtracted eig"stoxin
Th1roughout the twwnty yesr opan between tte Randall hypothesis jref 11)
wad the discovery of r, jqk.. the hoen of citoratr research was the lack
of toxin for research in Oweistry and Prrmeolosy It was sell recogittied
that the liver sand viscer% of the laige cartivoros, as P a moray **Is. proved
to be the richest ra Material for the extraction of cipaatmttn (rot. 23), but
-a thome sources contained toxis averaging no mere ithem WxO%. Then oneadds is this the formidable factors .f logistics end finances. spearing or
trapping the sole and transporti.-S them to a well-equippod laboratory. the
obotacles are inde dounti'g. Suddenly. those problems would be a thing of
the past. The discovery of the tosic ditnoflagelato g. jgxLQ&. the
dommstratuas that It elaboratoe$ the ether-seluble ciguaetxia and the
water-esoluble waltotextox (rof. 24). heralded a raw era In cigusterm research-
ample toxin would be available from dimeflacellate culture. There mesosd
PIP, det for this optimistic prognosis. other toxie marine dinotlagellats..notabiv rjW k 'opp. and 9ZJjdL" up.. had been successful ly cul tured for-ay year$.
The state of epotrmi&a es hart-l Ivic. first, the expoir lone. gained from
the cultuare of I ree-selaming dinoflse~llates did not readily oxtrepolale to
the benthic epiphytic Q. toucus. which could not be grown In large tanks nor
fouald its groth be onhaniced by aoration or agitation. Secondly, sand mre
oetimt..ly. early culture. produced, saitotoxin but only minimal traces of
cirunt*oni (ref. 20). Despite culturtag efforts in different laboratories
wver the past ten years this diseppointing situation has r mained e 1ssentially
I ahaged. Among recet experiences awe thmee of Drarpt-Cloment (ref. 25).
Wsh after considerable experimentation established cultures of G. AMU=I that
wore, oemaistently toxic sad produced sater-eoluble. mitotoxin-like. sand
liptd-eolublo. eigusatoin-lilte. fraction in ratio that varied from 9:1 to
7.6:2.5.
Nillerr anud coworkers (ref. 26) we"eee kila growing jm. C from aCaribbom collection a a mederately large scale, (20 liter carboys). An
aesmeent of the nautre of the texin@ derived from this culture to difficult
since the LDSw of the lipli extract after four chromatographic purification
ate" mes 4.15. ag/lu. or only 0.012 of the le~tholity of pure clguot~win.Their enter-soluble toxin men purified to an I.,of LI Wksla.
Csguatemin production from cultured g.j~gL reamine to be achieved.
There io sufficient circumstantial ovidence that genteti factors - existence
of nuontoxic strains of G. toxicus (ref. 10) - and environmental parameterspreferential settling on maeroalga. - play a role.
Product ion of meitotouttn tram taxijus cultures has bee* successful,
Good progress hats been wade towardi structural elucidation oi this toxin. which
is *pproxientely three times as large as cipatowein. Most if not all of this
hao been achieved in Professor Yasumte's laboratory. I will restrict this
269
account to the rtpatoxin stru~ture. which has beIn our concern for some time.
While the total structure is still unknown. Tachibana (ref. 27) isolated
chromatographically pure toxin with an LDSO of 0.45 pg/kg., This toxin
mubsequently mn seretdipitously crystallized in an NO tube enroute from Noe
Jersey to Hamwi. While attempts to determine a 13C MR1 spectrum had failed.
the crystalllnity of the toxin (Fig. 1) constituted additional and welcome
proof of its homogeneity. Extensive IH N spectral rnalyses of ciguatoxin at
GM 0 x (ret. 2) resulted in eet.blishwnt of the first unambiguous standard
of comparisne for all workers in the field.
.. 1MA,*
7 *
I ,,
Figure I. Crystals of cigtmtoxin from mst)mml. Approximate length. I mo.
While the mas spectral tectmology of the time did not allow high
Sresolution mas maaurements. a reliable moleclar lon at WS 1.111.7 f 0.3
dalton wms comatible with M U data. This number, combined with 114 MU
spectral interpretation, suggests C S9.HRNO1 9 as the most plausible molecular
formula of cigmltoxin.
Yasumoto's discovery that okadaic acid. which he had isolated from the
mrine dinoflagellate. Er.o rnntruM JIM (ref. 29). had the ,some
chromatographic mobility as ciguatoxin. placed t0e compound in the class of
polyethers. Since ciguatoxin possesses only five hydroxyl groupr, the bulk of
its oxygen atom must be part of -ther or kotal linkages. A comenrison of the
known structural .raemeters of citunto.in with those of okndaic acid (I).
first isolated from the sponge Lla r .0a Qý.ad (ref. 3)).
270)
norbmlichondrin-A (2) from the soam sponge (ref. 31). and prorocentrolide (3)
from P. JIM (ref. 32) i1 presented in Table 1. It is worth noting that
ciguatoxin is by far the most potent toxin among these four compounds and the
as yet unknown molecu ar architecture must account for this.
Me4 3-
oOHH2 0 2 1 24 e
Me MOH M@4 2 H 22 26 30 3I H Me'0
Uj1 E, HH H M H
;. 0 0•
3 1 30
0o .0 0
HO
me
3o.
* -
271
TABLU 1
Comparison of stru kural foatures and lethality of several dinoflagellatetoxins
Oludaic Acid Norhalichiondrin-A Prorocentrolide Ciguatoxin(1) (2) (3)
Sol..calim C4 4 H6 0 13 C5 9 HS2 02 1 C56HS5"03 C59HS0 1 9
Formula
Molecular Wt. 804 1.126 981 1.111Unsaturation 11 19 15 18 ÷:..
Chain Lenth 38 53 49No. of Rings 7 15 7
Methyls 5 4 5 5Exomethylwes 1 1 1 I
Hydroxyls 4 3 I 5
Ethers 1 7 3 ?
Acetal/Ketal 3 4 -?
, Carboxyl. Ester 1 2 1 ?
Other Functions 2 C C 5 C C. I C X 3C C
LDSO (mice) 0.19 q/lcg 0.4 uj/kS 0.45 mg/kg
The topics in the foregoing discussion - origin of the toxin.
dinoflagellate culture, molecular structure - are of fundamental scientific
Importance. In fact. niguatera Is an extraordinary ecological phenomenon that
opens trophic levels from unicellular alp. to man. But it oarm -re to poll a
cross section of people who are familiar with cigulatera - fish eaters.
fi'shmogers. fishermen, scientists - in an attempt to pinpoint that aspect of
ciguatera that is of greattst concern, no doubt the leading answer would be.
detection of the toxin. A random event that occurs witheot a tell-tale red
tide. that infects a broad spectrum of food fishes, that produces toxins which
are tasteless, odorless, and survive cooking. is. as was quoted in the
IlqTR(OXr•1Fl. an impressive type of fish poisoning. Hence development of a
rapid, simple, and inexpensive test that sounds the alarm for the first person
who hmndles the fish after it is caught and tells that person whether the fish
is safe to eat, would he)p the consumer who needs uncontaminated fish as well
as the scientist who needs the toxic fish for research. While the day of a
272
test that is comparable to the color change of litmus paper has not yet
arrived, great strides have been made toward this long sought-after goal.
iokama and coworkers (ref. 33) first described a radioim.munoessay (RIA)
for the detection' of ciguatoxin in 1977. In their assay ciguatoxin was
conjugated to human serum albumin and the resulting conjugate was injected
into s$:ep. Cigu•t•xin antibody produced by the sheep was purified and
coupled to radio-iodine (1251). The resulting radio-labelled antibody was
used successfully to screen more than 5.000 samples of ciguatera-prone
amberjack (Seriola dumerili. kagalj) prior to commercial sales in Hawaii
"between I97 and 1961 (ref. 34).
This ma a major breakthrough since previous bioassays either depended on
feeding a portion of a suspect fish to a susceptible animal (cat, mongoose) or
on lengthy extraction and partial purification of fish tissue, followed by
injection into a mouse. The RIA used small amounts of fish (10-20 pg) end no
longer depended on assay animals, but it still required a sophisticated
laboratory and a relatively long' (4 hours) experimental protocol. It was
clearly not an invention that was readily adaptable for use on a fishing boat
or in a fish market.
Hokam and coworkers (rrpf. 35) next turned to the development of an
enzyme imrnoessay (EIA). They coupled their sheep anti-ciguatoxin to
horseradish peroxidase. This conjugate plus a suitable marker could be
evaluated by measuring absortmne at 405 no in a spectrophotomer-. The new
"test proved to be simpler and faster, but still depended on instrumentation.
It also appeared that compounds that are structurally related to ciguatoxin.
as e.g. okmdati acid. would also bind to the sheep, anti-ciguatoxin. This is
not necessarily a drawback. since okadaic acid. though less toxic. may well be
present in fish and contribute to the toxic syndrome.
The 'third stage of the quest for a ciguatera detection test led Hokasma
(ref. 38) to his socalled stick toet, in which a coated bamboo skewer is-inserted into the fish. The stick is then rapidly prepared for insertion into
the test (i.e. sheep anti-ciguatoxin horseradish peroxidase) solution and
visually evaluated. While this test is still- not wholly specific and the
visual evaluLtion of a color involves subjective factors, it is rapid.
inexpensive, and can be used in the field.
THERAPY
The success achieved in the long quest for a rapid, simple, inexpensive
test for ciguatoxic fish. described in the previous section. was the result of
a deliberate research program based on immunological techniques and spanning
more than a decade. Another unsolved ciguatera problem, what to do once
273
Intoxlcation has occLrred. has apparently also found a solution. In contrast
to the carefully charted and executed program toward development of the stick
test, the newly suggested ciguatera treatment (ref. 37) ms an empirical
discovery. Its importance to the people who live in endemic ciguatera regions
is underlined by the fact that the description of the therapy (ref. 37) was
preceded by P notice in the lay press (ref. 38) and was folllowed by an
editorial in a Honolulu newspaper (Fig. 2). The highly effective use of
Miracle drugDD mannitol to the list of miracle
drugs along with aspirin and Denicil-lin. Mavnitol, commonly used to re-
duce brain swelling, is an artificially pro-duced sugar compound that has been foundto banish permanently all effects of the fishpoisoning ciguatera.
The antidote, discovered and tested by Uni-versity of Hawaii physicians, has been usedon 40 seafood eaters who were seriously Illfrom the poisoning. The affliction is commonthroughout the Pacific, in Florida and the Ca-ribbean. Ciguatera poisoning is rarely fatal,but its symptoms - numbness, vomiting anddiarrhea - make victims miserable and cancause unconsciousness.
Dr. Neal Palafox, a graduate of the UHJohn A. Burns School of Medicine practicingin the Marshall Islands, is credited with dis-covering mannitol's effect on ciguatera pa-tients.
Although the drug hasn't been used in Ha-waii, there is no reason to doubt it wouldn'tperform just as well here. Seafood lovers caneat a little easier now
Fig. 2 Honolulu Star-Bulletin. June 6. 198
L/
274
intravenous infusion of D-mannitol (4) with drammtic results was foreshadowedby similar, though ineffective, calcium gluconate therapy in Fiji (ref. 39).The very sach lower dose of calcium gluconate Infusion my well account forIts failure at the time. Both gluconic acid (5) and mannitol are simplehexose derivatives which differ in only two of the six carbons. Thiscoincidence my prove to be of theoretical interest once the structures ofciguataxin and mattotoxin are known. Computer modelling studies may then shedlight on the mechanism by which 'intoxication takes place.
-. -HD- -- H H- -"4n.
HD- -HI- ---
'- -- O- H--O
H- --- If- ---%CH %OH4 5
(CqCL3SI(
The decade since the discovery of Gm-bierdisczs t has seen
impressive progress in ciguatera research: G. toxicus is being cultured and
the cultures are producing maitotoxin; ciguatoxin has been spectrally
characterized and has been obtained as a crystalline compound: a simple.
rapid. inexpensive test is at band; and an empirical therapy appears to b.,
effective. Still poorly understood are the ecology of G. toicus; the factors
- genetic and/or environmental - that govern cigetoxin biosynthesis in wild
and In cultured populations: and the interrelationship of ciguatoxin and
miltotaxin at all trophic levels.
1 S.K. Sutherland. What on earth is ciguatera, fed. .1. Au ,trall 145(.1/12)(19w6) 558-559.
2 N.C. Cillespie. R.J. Lewis. J.H. Pearn. A.T.C. Bourke. M.J. Holmes. J.B.Bourke and W.J. Shields, Ciguatera in Australia, e. J. Australia145(ll/12)(1996) S54-590.
3 E.P. Ragells (Ed.). Seafood toxins. ACS Symposium Series 262. AmericanChemical Society. Washington. DC. 1984. pp. 217-329.
4 T. Higerd. D. Ballantine. D. Jollow and P. Scheuer (Eds). The secondinternational conference on ciguatera, Marine Fish. Rev. 48(4) (1996) 1-59.
5 R. Bagnis and P:J. Scheuer (Eds.) Ciguatera and other reef seafoodpoisoning, in: Proceedings of the Fifth International Coral Reef Congress.Tahiti. 1985. Vol. 4. Antenne Museum-Ephe. Moorea. French Polynesia. 1985.pp. 401-502.
6 First International Workshop in Ciguatera. Beverly. MA. 1995: Biol. Bull.172(1) (19S7) pp. 89-153.
,/
275
7 N. Nukina. K. Tachibana and P.J. Scheuer. Recent developments in ciguateraresearch. in: J.B Harris (Ed.), Natural toxins. Clarendon Press. Oxford.UK. 1986. pp. 46-55.
8 Y. Hiywma, Report of an investigation on poisonous fishes in the south seas(-n Japanese), Nissan Fisheries Institute. Odawara. 1943. [Englisht.ranslation: W.C. van Ceapen. Special Scientific Report - Fisheries. No.25, U.S. Fish and Wildlife Service. 1950. pp. 1-221.)
9 Y. Hashimoto. A note on the poison of a I-arracuda. Sphyrae picuda Bloch &Schneider. Bull. Jap. Soc. Sci. Fish. 21(11) (1958) 1153-1157.
10 B.W. lilstead, Poisonous and venomous mrine animals of the world. U.S.Gov -rnment Printing Office. Washington. DC, 19t5. 1967. 1970.
11 .I.E. Randall. A review of ciguatera. tropical fish poisoning, with Ptentative explanation of its cause. Bull. Marine Sci. Gulf Carib. 8(3)
) (1968). 236-267.12 P. Helfrich. a. Piyakarnchanm and P.S. Miles. Ciguatera fish poisoning I. . -.
The ecolory of ciguateric reef fish in the Line Islands, Occasional Papersof Bernice P. Bishop Museum. Honolulu, HI. 23(14) (1968), pp. 305-382.
13'T. Yasumoto, Y. Hashimoto. R. Bagnis. J.E. Randall and A.H. Banner.Toxicity of surgeonfishes. Bull. Jap. Soc. Sci. Fish 37(8) (1971) 724-734.
14 T. Yasumoto, I. Nakajira. R. Bagnis and R. Adachi, Finding of adinoflagellate as a likely culprit of ciguatera. Bull. Jap. Soc. Sci. Fish.43(8) (19T7). 1021-1026.
15 R. Adachi and Y. Fukuyo. The thecal structure of a marine toxicdinoflagellate Cambierdiscus t gen. et sp. nov. collected in aciguatera-endemic area. Bull. Jap. Soc. Sci. Fish. 45(1) (1979). 67-71.
16 T. Yasumoto. Recent progress in the chemistry of dinoflagellate toxins, in:D.M. Anderson. A.W. White and D.C. Baden (Eds.) Toxic dinoflagellates,Elsevier, New York. 1985. pp. 259-270.
17 T. Yasumoto. R. Bagnis and J.P. Vernoux. Toxicity of the surgeonfishes 2.Properties of the principal water-soluble toxin. Bull. Jap. Soc. Sci. Fish.42(3) (1976). 359-365.
18 N. Gillespie. R. Lewis. J. Burke and M. Holmes. The significance of theabsence of ciguatoxin in a wild population of g. toxicus, in: Proceedingsof the Fifth International C•oral Reef Congress. Tahiti. 1965, Vol. 4.Antenme Museum-Ephe. Moorea. French Polynesia. 1965, pp. 437-441.
19 Y. Shimizu, H. Shimizu, P.J. Scheuer, Y. Hoklam. M. Oyau and J.T.Miyahara. Gambierdiscus toxicus. a ciguatera-causing dinoflagellate fromHawaii. Bull. Jap. Soc. Sci. Fish. 48(6) (1982). 811-813.
20 T. Yasumoto. K. Fujimoto. Y. Oshims. A. Inoue. T. Ochi. R. Adachi and Y.Fukcyo. Ecological and distributional studies on a toxic dinoflagellateresponsible for :iguatera. Report to the Ministry of Education. Tokyo.Japer, 199.. 50 pp.
21 T. Ytsumoto. A. Inoue. R. Bagnis and N. Garcon. Ecological survey on a3 dinoflagellate possibly responsible for the induction of ciguatera. Bull.Jap. Soc. Sci. Fish. 45 (3) (1979). 396-399.
22 J.P. McMillan, P.A. Hoffman and J.R. Grenade. bierdscs toxicus fromthe Caribbean: A source of toxins involved in ciguatera. Marine Fish. Rev.48(4) (1996). 48-52
23 T. Yasumoto and P.J. Scheuer, Marine toxins of the Pacific - 7-Ciguatoxinfrom moray eel livers. Toxicon 7(4) (1969). 273-276.
24 R. Bagnis. S. Chanteau. E. Chungue. J.M: Hurtel. T. Yasumoto and A. Inoue,Origins of ciguatera fish poisoning: a new dinoflagellate. Ckimbierdiscustoxicus Adachi and Fukuyo. definitely involved as a causal agent. Toxicon18(2) (1980). 199-208.
25 M. Durant-Clement. Stuiv of production and toxicity of culturedCambierdiscus toi.icus, Bin.. Bull. 172(l), (19•7). 108-121.
26 D.M. Miller. R.W. Dickey and D.R. Tindall. Lipid-extracted toxins from adinoflagellate. Qambierdiscus toxicus. in: E.P. Ragelis (Ed.). SeafoodToxins. ACS Symposium Series No. 262. American Chemical Society.Washington. D.C. 19S4. pp. 241-255.
276
27 K. Tachibana. Structural studies on marine toxins, Ph.D. Dissertation.University of Hawaii. Honolulu. HI. 1980.
/ 28 M. Nukina. L.N. Koyanagi and P.J. Scheuer. Two interchangeable forms ofciguatoxin. Toxicon 22(2) (1984). 169-176.
29 Y. Nuraksmi. Y. Oshims and T. Yasumoto. Identification of okadaic acid as atoxic c o t of a marine dinoflagellate. Prorocent uLr. Bull. jap.Soc. Set. Fish. 48(1) (1982). 69-72.
30 K. Tachlbana, P.J. Scheuer. Y. 'sukltani. H. Kikuchi. D. Van Engen, J.Clardy, Y. Gopichand and F.J. Schmltz. Okadaic acid. a cytotoxic polyetherfrom two marine sponges of the genus Halichondria J. As. Chem. Soc. 103(9) (1981). 2469-2471.
31 D. Ueinzra. T. Takahashi. T. Yamamoto. C. Katayrma. J. Tanaka. Y. Okamraand Y. Hirata. Norhalichondrin A: an antitumor polyether macrolide from amarine sponge. J. Am. Chem. Soc. 107(16) (1985). 4796-4798.
32 K. Torigoe. N. Murata and T. Yasumoto. 'Prorocentrolide. a novel polyethermine from the marins dinoflagellate. .rorocent Jlm, Abstracts. 16thInternational symposium on the Chemistry of Natural Products (IUPAC). **• ....
May 29 - June 3. 1988. Kyoto. Japan. Pa 20.33 Y. Hokams. A.H. Banner and D.B. Boylan. A radioinnmoassay for the *_.
detection of ciguatoxin, Toxicon. 15(4) (1977). 317-325.34 L.H. Kimara. N.A. Abed and Y. Hokama. Evaluation of the radioimmunoassay
(RIA) for detection of ciguatoxin (CTX) in fish tissues. J. Fish Biol.21(6) (;98). 671-680.
35 Y. Hokama. N.A. Abed and L.H. Kimura. A rapid enzyme imanoassay (EIA) forthe detection of ciguatoxin in contaminated fish tissues. Toxicon 21(6)(198). 817-824.
36 Y. Hoklma. A rapid, simplified enzyme iinmosay stick test for thedetection of ciguatoxin and related polyethers from fisn tismues. Toxicon23(6) (1985) 939-946.
37 N.A. Palafox. L.G. Jain. A.Z. Pirano, T.M. Culick. R.K. Williams and 1.J.Schatz. Successful treatment of ciguatera fish poisoning with intravenouswannitol. J. Am. Ned. Assoc. 259(18) (1988). 2740-2742.
38 G. Johnson. Marshalls' M.D. curing fish poison. Pacific Magazine 13(2)(1988). 51.
39 N. Soroklin, Ciguatera poisoning in north-west Viti Levu. FiJi Islands, Haw.Ned. J. 34(6) (1975). 207-210..-
*.] I'.
I