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AD____

MARINE BIOTOXINS: LABORATORY CULTUREAND MOLECULAR STRUCTURE

I% uAnnual Report

Paul J. Scheuer, Ph.D.

October 18, 1989

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U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick# Frederick. Maryland 21701-5012

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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 . .

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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

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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.

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