The Chemical Nature of Scaritoxin

YONG-GOE JOH and PAUL J. SCHEUER

Introduction

One of the many questions associatedwith ciguatera research has been that ofthe multiple nature of the toxins thatgive rise to ciguatera symptoms in man.The lipophilic ciguatoxin (Scheuer etal., 1967) and the hydrophilic maito­toxin (Yasumoto et al., 1976) are distinctand reasonably well characterized mole­cules, though their specific contribu­tions to the ciguatera syndrome have notbeen assessed.

Bagnis et al. (1974) conducted an epi­demiological survey of ciguatera intox­ication in the Gambier islands andobserved that the most frequently im­plicated fish were parrotfish (Scaridae).Afflicted persons would, in addition tothe conventional immediate ciguaterasymptoms, suffer significantly fromdelayed (by 5-10 days) and prolonged (1or more months) episodes of disturbedequilibrium, locomotor difficulties, andkinetic tremors.

An obvious explanation is the pres­ence in parrotfish of a new toxin calledscaritoxin, or of two distinct toxic en­tities (Chungue et al., 1977a, b). Fromthe flesh of Scarus gibbus, Chungue etal. (1977a, b) successfully separated twotoxins by chromatography on DEAE

ABSTRACT-Two toxins were isokitedfromScams sordidus (Family Scaridae) collectedat Tarawa atoll, Republic of Kiribati. Bothtoxins were present in the flesh and viscera,but in different ratios. They correspond tothe previously described scaritoxin andciguatoxin. The toxins can be interconvertedand evoke parallel symptoms in mice. Scari­toxin is probably identical with less polarciguatoxin, but lack of material preventedunequivocal proofby spectral comparison.

48(4), 1986

cellulose. A toxin eluted with chloro­form and designated SG-l evoked slug­gishness and severe hind limb paralysisin mice, different from typical cigua­toxin symptoms. A second toxin elutedwith chloroform-methanol (1:1) anddesignated SG-2 produced conventionalciguatoxin symptoms in mice, such asdiarrhea, lachrymation, salivation, andrespiratory difficulties.

Both toxin entities were further puri­fied on Sephadex LH-20. SG-l was ayellowish oil with an LDso of 0.03 mg/kg. The polar toxin SG-2 was comparedchromatographically with moray eelciguatoxin and was found to be indistin­guishable. Both toxins, SG-l and SG-2,responded negatively to reaction withiodine and to Dragendorff and Jaffereagents. A chromatographic estimate ofthe molecular weight of the new, lesspolar SG-l toxin was approximately 800daltons.

Chungue (1977) in a detailed investi­gation substantiated these results for sixadditional species of parrotfish. Sheconcluded that scaritoxin is a ciguatera­causing toxin that is characteristic of theScaridae.

In a more recent study, Nukina et al.(1984) showed that ciguatoxin isolatedfrom moray eel, Lycodontis javanicus(= Gymnothorax), viscera can be sep­arated on basic alumina into two en­tities, differing in polarity but indistin­guishable in symptoms or in LDso (i.p.mice). The two toxins show only minordifferences in their high field 'H NMFspectra. It was further shown that thetwo toxins are interconvertible. It oc-

The authors are with the Department of Chemistry,University of Hawaii at Manoa, 2545 The Mall,Honolulu, HI 96822. The pennanent address ofYong-Goe Joh is: Department of Food Science andNutrition, Dong-A University, Pusan, Korea.

curred to us that these two chromato­graphic forms of ciguatoxin might beidentical with the scaritoxin-ciguatoxinpair recognized in the flesh of parrot­fish by Chungue (1977). We had an op­portunity to collect parrotfish, S. sor­didus, on Tarawa atoll, Republic ofKiribati, and thus subject this hypoth­esis to an experimental test.

Materials and Methods

Fish were collected 19-17 May 1983at Taborio, Betio, Bikenibeu, and Tea­oraereke (Fig. 1). Small-scale extractionand testing showed that only S. sordiduscaught at Bikenibeu were toxic. Flesh(5.34 kg) and viscera (0.876 kg) wereworked up separately.

Fish flesh was mixed with acetoneand reduced to a brei in a I-gallon War­ing Blendor'. The mixture was trans­ferred to a 4 liter Erlenmeyer flask andallowed to stand in excess acetone for2 days, then with fresh acetone foranother 4 days. The combined acetoneextracts were concentrated to an aque­ous suspension, which was treated twicewith equal volumes of hexane. The hex­ane layer was back-washed with 3 X 100rn1 of methanol/water, 8:2. The wash­ings were added to the aqueous suspen­sion, and the combined aqueous materi­al was extracted three times with equalvolumes of ethyl acetate. Evaporation ofethyl acetate resulted in crude toxin.

Crude toxin was dissolved in a mini­mum amount of chloroform and appliedto the top of a column packed withSilicar (200-425 mesh, Mallinckrodt,28 gig sample). Successive elution withchloroform (10 ml/g silica), chloroform!

IReference to trade names or commercial finnsdoes not imply endorsement by the NationalMarine Fisheries Service, NOAA.

19

( ) Sand covered reef, mainly dry at low tide~----~

- - - - - - - - - - - - - - Approximate edge of lagoon

The toxic fractions (polar toxin, 450ng; less polar toxin (810 ng) were fur­ther purified on a Sephadex LH 20(Pharmacia) column (103 x 1.4 cm) byelution with cWoroformlmethanol (2:1).Fractions (4.9 ml) were collected every10 minutes. Elution was monitored at254 nm (ISCa Model UA-5).

Aluminum plates coated with silicagel 60F-254 (0.2 mm) and glass platesspread with silica gel H were used forthin-layer chromatography (TLC).Plates were developed with cWoroformlmethanol/water/acetic acid (90:9.5:0.3:0.2) and visualized with iodine vapor.Spots or bands were scraped off and ex­tracted with chloroform/methanol, 4:1for the less polar and 1:1 for the polartoxin. For bioassay the recovered TLCfractions were homogenized in 1 percentTween 80.

For bioassay, each sample was dis­solved in a known volume of methanol.Aliquots were removed from this stocksolution and solvent was removed by astream of nitrogen. Tween 80 (0.5 mlof 1 percent or 0.1 ml of 5 percent) wasadded and homogenized in a Vortexmixer. Swiss Webster mice (male orfemale, 16-22 g) were injected intraperi­toneally. Death times of less than 3hours were used to estimate toxicity asdescribed by Tachibana (1980).

173"10'

~-N-

~

173"05'

SCAlE

L-'9_-'----'1_--'----"----'STAIUIE MILES

o ,

"

(~ ~) Dry land above sea level

17'5trJ'

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~ :.'~~TABORIO

I;::> , ~ ••~TEAORAEREKE

172"55'

,I,,

(I' ~t::~~··; t:\,,\II\,I

II

Figure I.-Tarawa atoll, Republic of Kiribati.

methanol (9:1, 14 ml/g silica), chloro­form/methanol (1:1, 10 ml/g silica), andmethanol (10 ml/g silica) separated thetoxin. All fractions were evaporated todryness immediately after elution.

The chloroform/methanol fraction(9:1) was further purified on a DEAEcelulose column (Cellex D, acetateform, BioRad Laboratories, 9.3 gigsample), which was prepared as previ­ously described (Rouser et aI., 1963).Eluting solvents were chloroform (60ml/g adsorbent), chloroform/methanol(1:1, 90 ml/g adsorbent), and methanol(60 ml/g adsorbent). Guaiazulene (ablue pigment, Aldrich Chemical Co.)was used to determine the bed volumeand to detect imperfections in the pack­ing.

An alumina (activity grade I, Woelm)column was prepared by suspendingalumina (100 gig sample) in cWoroform.Toxic fractions were added as chloro­form solutions and were eluted succes­sively with chloroform/methanol (100:1, 9:1, 1:1), methanol, methanol/water(1:1), TI5 ml/g alumina.

Alumina activity grade V was pre­pared according to the Brockmannscale. Grade I and V alumina columns(18.5 X 0.7 cm) were packed with 10 galumina each and the toxins were elutedwith chloroform (40 ml), chloroformlmethanol (36 + 4 ml), chloroformlmethanol (1:1), methanol (40 ml), andmethanol/water (1:1). The eluates wereconcentrated and the residues weredissolved.

Results

Small-scale extractions and bioassayshowed that S. sordidus was the onlytoxic species; S. jrenatus, S. scaber, andS. pectoralis were nontoxic.

Yields and lethalities of extracts andchromatographic fractions are shown inTables 1 (flesh) and 2 (viscera). Theviscera proved to have a higher concen­tration of toxin, a phenomenon that hadpreviously been observed in the morayeel (Yasumoto and Scheuer, 1969). Tox­in could be eluted from silicic acidcleanly in cWoroform/methanol (9:1).The toxin was separable into polar andless polar entities on DEAE cellulose.In S. sordidus flesh the polar toxin pre­dominated, while in the viscera thereverse was so. When the chloroformlmethanol (1:1) fraction from the DEAEcellulose column was rechromato­graphed on alumina activity grade I and

20 Marine Fisheries Review

Figure 2.-Interconversion of Scarus sordidus flesh toxins on alumina.

I After treatment with IN NaOH in aqueous methanol.

28: chloroform-methanol (9:1). C: chloroform-methanol (1:1).E: methanol-water ( I: I ).

Column charge ST-I,I.I~g ST_II, 1.13 ~g ST-2, I. 2 ~g

Activity grade I I II.

Eluates 2I I I I I I I I I

~I I

8 C E 8 8 EAmounts

0.12 0 . 15of toxin 0.55 0.12 0.34 0.21 0.80recovered ~g ~g ~g ~g ~g ~g ~g

Table 3.-Comparlson of Rr-values of S. 50r­d/dus flesh toxins with PCTX (Tachlbana, 1980)and scaritoxln (Chungue, 1977). Aluminumplates were coated with silica gel 60F·254 (0.2mm): solvent system was chloroform/methanol!water/acetic acid (90:95:0.2:0.3).

to ST-1 by chromatography over deac­tivated (Grade V, 15 percent water)alumina. When ST-1 is first treated withaqueous methanolic sodium hydroxide,then chromatographed on alumina of ac­tivity I, partial conversion to ST-2 alsotakes place, but the loss of material isnecessarily severe. Paucity of availabletoxin prevented us from carrying out afull-scale experiment. Results are shownin Figure 2. The less polar toxin isdesignated ST-l, the polar toxin, ST-2.

On subsequent chromatography onSephadex LH 20, ST-l and ST-2 areeluted in parallel fractions (88-110 ml forST-1 and 86-108 ml for ST-2) , thusdemonstrating the identical size of thetwo toxins.

To show the relationship of the twoS. sordidus toxins to ciguatoxin (Nukinaet al., 1984) and to scaritoxin (Chungue,1'177), we carried out thin layer chroma­tography under Chungue's conditions.Within the normal variability of TLCwith time and place, and with a four­solvent developer, ST-1 and scaritoxinare very likely to be identical, as areST-2 and ciguatoxin (Table 3). The TLCspots are egg-shaped rather than cir­cular. Significantly, though, the Rrvalues do not overlap. Amounts of toxinsufficiently large for 'H NMF com­parison are needed for unequivocalproof.

21

Discussion

The demonstration of two chromato­graphically distinct toxins in a cigua­toxic fish, while interesting by itself,raised another question concerning theprecursor(s) of the toxins. Parrotfish, S.gibbus, feed primarily on coral. To shedlight on the origin of the toxin, Yasu­moto in collaboration with workers inTahiti (Yasumoto et aI., 1'177) examinedthe gut contents and liver of S. gibbus.The gut, surprisingly, contained noscaritoxin, but contained ciguatoxin andmaitotoxin in addition to a fast-actingacetone-soluble paralysis-causing toxin.Only ciguatoxin was isolated from theliver. Since both ciguatoxin and scari­toxin are routinely isolated from flesh,these results suggest that the parrotfishmay have the ability to transform cigua­toxin into scaritoxin, yet the absence ofscaritoxin in the liver was puzzling.

0.92

0.78-

ScaritoxinPCTX

0.28-

0.54

ST-2

0.54

0.30-

ST-1

0.60-

0.75

eluted with a gradient beginning withchloroform/MeOH (100:1) and endingwith methanol/water (1:1),95.5 percenttoxicity was eluted with methanol/water(1:1). The two toxins gave rise to paral­lel symptoms in mice.

In analogy with the recent demonstra­tion (Nukina et al., 1984) that ciguatoxinfrom moray eel viscera can be separatedinto two distict entities of differentpolarity by alimina chromatography andthat the two toxins are interconvertible,we were able to show that the two S.sordidus toxins obtained by DEAEcellulose chromotography can also bepartially interconverted. Figure 2 showsthat ST-1 is partially converted to ST-2when passed through a column of highlyactive (Grade I) alumina. ST-2, on theother hand, can be partially converted

Table 1.-Yields and toxicity of extracts and of chrom-atographic fractions of S. sordidu5 flesh (5.34 kg).

TotalYield LDso toxicity

Purification state (g) (mg/kg) (M.U.)

Ethyl acetate 8.99

Silicic acidChloroform 3.52 NontoxicChloroform/methanol 1.53 23.0 3,319(9:1)(1:1) (9:1) 0.63 12.5 2,520

(1:1) 1.13 NontoxicMethanol 2.20 Nontoxic

DEAE-celluloseChloroform 2.74 29.0 556Chloroform/methanol 0.52 22.5 1,160(1:1)Methanol 0.01 Nontoxic

Table 2.-Ylelds of extracts and of chromatographicfractions of S. 50rdidu5 viscera (0.876 kg).

TotalYield LDso toxicity

Purification state (g) (mg/kg) (M.U.)

Ethyl acetate 13.81

Silicic acidCHCI3 4.46 NontoxicCHCI,.CH3OH 5.05 29 8,763(9:1)CHCI,.CH,oH 1.48 Nontoxic(1:1)CH,oH 1.58 Nontoxic

DEAE-celluloseCHCI3 2.94 29.5 5,034CHCI,.CH,oH 1.00 27.8 1,799CH,oH 0.08 Nontoxic

48(4), 1986

Our findings that toxic parrotfishfrom Tarawa atoll come from a narrowgeographic area (Fig. 1) is consistentwith the traditional ciguatera pheno­menon (e.g., Withers, 1982). We wereboth surprised, though, that of fourspecies examined, only S. sordidusproved toxic. We were able to isolate twotoxins, parallel with Chungue's (1fJ77)work on S. gibbus. In contrast to Yasu­moto's (Yasumoto et al., IfJ77) results,we were able to show that both toxinsare present in the flesh and in the vis­cera, albeit in different proportions. Weobserved no evidence of a third acetone­soluble and fact-acting toxin (Yasumotoet al., IfJ77). This is not a critical point,as coral-feeding parrotfish have manyopportunities to ingest other toxins.Bagnis' (1fJ74) original observation ofthe different symptomology in man

22

following intoxication by parrotfish re­mains unexplained.

Acknowledgments

We thank Mark R. Hagadone andDominic McCarthy for collections, JohnE. Randall for identification of fishes,and the National Marine Fisheries Ser­vice (NA80AA-OOlOl) through a sub­contract with the Medical University ofSouth Carolina for financial support.

Literature Cited

Bagnis, R .• E. Loussan, and S. Thevenin. 1974.Les intoxications par poissons perroquets auxIles Gambier. Med. Trop. 34:523-527.

Chungue, E. 1977. Le complexe toxinique despoissons perroquets. Ph.D. thesis. Academiede Montpelier, Universite de Sciences et Tech­niques de Languedoc, Montpelier, France.

____ , R. Bagnis, N. Fusetani, and Y.Hashimoto. 1977a. Isolation of two toxins froma parrotfish Scarus gibbus. Toxicon 15: 89-93.

, and T. Yasumoto,

1977b. Le complexe toxinique des poissonsperroquets. Biochimie 59:739-741.

Nukina, M., L. Koyanagi, and P. 1. Scheuer. 1984.Two interchangeable forms of ciguatoxin. Tox­icon 22:169-176.

Rouser, G., G. Kritchevsky, D. Heller, and E.Lieber. 1963. Lipid composition of beef brain,beef liver, and the sea anemone: Two ap­proaches to quantitative fractionation of com­plex lipid mixtures. 1. Am. Oil Chern. Soc.40:425-454.

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

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

Withers, N. W. 1982 Ciguatera fish poisoning.Ann. Rev. Med. 33:97-111.

Yasumoto, T., R. Bagnis, and J. P. Vernoux. 1976.Toxicity of surgeonfishes. - II. Properties ofthe principal water soluble toxin. Bull. Jpn. Soc.Sci. Fish. 43:359-365.

----,=-----,-_ , I. Nakajima, E. Chungue, and R.Bagnis. 1977. Toxins in the gut contents ofparrotfish. Bull. Jpn. Soc. Sci. Fish. 43:69-74.

_,------,--,- , and P. 1. Scheuer. 1969. Marine tox­ins of the Pacific - VIII. Ciguatoxin from morayeel livers. Toxicon 7:273-276.

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