A tetrodotoxin-like substance as a minor toxin in the xanthid crab Atergatis floridus

Torfron, Vol. 22, No. 3, pp.425-432, 1984. Printed in Great acitain .

0041-0101/8453 .00+ .00 © 1984 Perpenon Peen Ltd .

A TETRODOTOXIN-LIKE SUBSTANCE AS A MINOR TOXIN IN THE XANTHID CRAB ATERGATIS FLORIDUS UZU,' KINDE DAI00 (KOYAMA),' YASUO SHIDA= and KANEHISA HASHIMOTO'* 'Laboratory of Marine Biochemistry, Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, and rI'okyo College of Pharmacy, Hachioji, Tokyo 192-03, Japan TAMAO NOGUCHI,' ATSUSHI

(Acceptedfor publication 3 January 1984) T. NoGUCEU, A. U2,u, K. DAIGO (KOYAMA), Y . $H1DA and K. H~+sl~ul~toro . A tetrodotoxin-like substance as a minor tOx1II in the xanthid crab Atergatisfioridus . Tiaxirnn 22, 425 -432, 1984 . Toxins were extracted from the xanthid crab Atergatls jloridus inhabiting Iahigaki Island, Okinawa, and subjected to several types of chromatography, resulting in separation into gonyautoxin and saxitoxin fractions. Thin-layer chromatographic and electrophoretic analyses showed that the gonyautoxin fraction wax composed of gonysutoxins 1-4, along with some unknown compounds. Cias chromatography-mass spectrometry demonstrated that the gonyautoxin fraction gave rise to the G-base when alkali-hydrolyzed, indicating that this fraction contained a tetrodotoxin-like compound possessing the gnina~nline skeleton specific to tetrodotoxin . The saxitoxin fraction consisted of neosaxitoxin, saxitoxin and two unknown compounds. INTRODUCTION

that three species of xanthid crabs, Zosimus aeneus, Atergatis}ïoridus and Platypodia granulosa, inhabiting Ishigaki Island, Okinawa, contain paralytic shellfish poisons such as saxitoxin (STS and neosaxitoxin (neoST~ (HASHIMOTO et al., 1969; NOGUCHI et al., 1969; KOYAMA et al., 1981). The toxin compositions of the three crabs differed from one to another. YASUMOTO et al. (1981) reported similar results. We recently examined the toxin composition of A. floridus specimens collected from Miura Peninsula, Kanagawa Prefecture, and found that they contained tetrodotoxin frTx) as the major component, along with small amounts of paralytic shellfish poisons (NOGUCHI, et al., 1983). With these new findings in mind, we re-examined the toxin composition of A. floridus crabs from Ishigaki Island and detected a TTX-like substance, in addition to paralytic shellfish poison components thus far reported . WE REPORTED

MATERIALS AND METHODS Materials Thirteen specimens of Atagatis floridus (average body weight, 15 .6 g) were collected on the coral reefs at Kabira, Ishigaki Island, Okinawa, in April 1983 . The specimens were immediately frozen and transported to the Laboratory of Marine Biochemistry, and kept frozen at -20°C until used . Purjficatlon ojtoxln Appendages and carapaces of the crab specimens were minced in a mortar . To the mince (95 g) wax added 300 ml of 805 ethanol addified to pH 2 .0 with HCI, and the mixture wax homogenized and filtered . The above procedures were reputed twice on the residue. The filtrates were combined, concentrated under reduced pressure and defatted with chloroform . The extract (total lethality, 88,000 MU) wax concentrated to 50 ml and "To whom correspondence should be addressed. 425

426

T. NOGUCHI et al .

applied to a column (2 .5 x 29 cm) of Amberlite IRC-50 (NH,' form) (Rohm 8c Haas, Philadelphia, PA, U.S .A.) . The column was washed with 1500 ml of water and developed with 10% acetic acid . The toxic fractions, monitored by mouse assay, were combined and freeze-dried . The solid was dissolved in water and chromatographed on a column (6.0 x 55 cm) of Bio-Gel P-2 (Bio-Rad Laboratories, Richmond, CA, U.S .A .) . The column was washed with water followed by 0.1 N acetic acid, and the toxic fractions were combined and freeze-dried . The toxin thus obtained was dissolved in water and rechromatographed on Bio-Gel P-2 in the same manner as described above. Thetoxic fractions were combined and lyophilized. The lyophilizate was dissolved in a small amount of water and subjected to chromatography on a column (0.8 x 95 .5 cm) of Bio-Rex 70 (H' form) (Bio-Rad Laboratories), by a two-step linear gradient using 0-0.03 N acetic acid and then 0.03-1.5 N acetic acid . The toxic fractions were monitored by mouse assay. Two toxic peaks appeared, one being eluted with 0-0.03 N acetic acid and the other with 0.03-1.5 N acetic acid. Fractions comprising each peak were combined, freeze-dried and analyzed as described below. Gonysutoxins (GTXs), STX and TTX were prepared according to the methods reported previously (Ut:nn et al., 1982 ; Noaucxi et al., 1969, 1981) and used as authentic specimens. Assay of lethal potency Unless otherwise specified, lethality was assayed by the method for paralytic shellfish poison (KAWABATA, 197ßa) and expressed in mouse units. One mouse unit (MiJ) is the amount of toxin required to kill a 20-g mouse in 15 min. In the assay method for TTX (KAWAHATA, 197ßb), one mouse unit is defined as the amount of toxin required to kill a 20-g mouse in 30 min. Thin-layer chromatography Thin-layer chromatography was performed on 5 x 20 cm silica gel 60 precoated plates (E . Merck, Darmstadt, F.R .G .) with a solvent system of t-butanol-acetic acid-water (2 :1 :1) or on 10 x 10 cm LHP-K high-performance precoated plates (Whatman, Clifton, NJ, U.S .A .) with a solvent system of pyridine-ethylacetate-acetic acid-water (15:5 :3 :4). Toxins were visualized as a yellow or blue fluorescent spot under u.v . light (365 nm) after spraying the plate with 1% H,O, and 10% KOH followed by heating at 110°C for 10 min (paralytic shellfish poisons and TTX) or visualized as a pink spot by spraying the plate with Weber reagent (TTX). Electrophoresis Electrophoresis was conducted on 5 x 18 cm cellulose acetate strips (Chemetron, Milan, Italy) in 0.08 M Tris - HCI buffer (pH 8.7) under a constant current of 0.8 mA/cm width for 60 min. Toxins were detected with 1% H,O, and 10% KOH, as in thin-layer chromatography. Gas chromatography-mass spectrometry (GC-MSJ The C9 -base, 2-amino-6-hydroxymethyl-8-hydroxyquittazoline, was derived from the authentic specimen of TTX by alkaline hydrolysis, and trimethylsilylated (Nnxrrn et al ., 1981). A portion of the toxic fraction (GTX fraction) eluted from the Bio-Rex 70 column with 0-0.03 N acetic acid was similarly alkali-hydrolyzed and trimethylsilylated . Both trimethylsilyl derivatives thus obtained were analyzed with a Hitachi GC-mass spectrometer M-80 . A column (0 .3 x 200 cm) of Chromosorb W coated with 1 .5% OV-101 was used and the temperature raised from 160°C to about 240°C at a rate of 5°C/min. The flow rate of inlet helium carrier gas was maintained at 20 mU min. The ionizing voltage was kept at 70 eV and the ion source temperature at 200°C. Scanning was carried out over a mass range of m/z 33 - 800 at 8-sec intervals.

RESULTS

Purification of toxin A total of 88,000 MU of toxin was extracted from the A. fioridus homogenate and purified by chromatography on Amberlite IRC-50 and Bio-Gel P-2. The toxin thus obtained (54,000 MU) was subjected to Bio-Rex 70 column chromatography . Its elution profile is shown in Fig. 1 . Most of the lethal potency (48,000 MU) was eluted with 0.03 -1 .5 N acetic acid (STX fraction), while only a small portion of lethal potency (2,200 MU) was eluted with 0-0.03 N acetic acid (GTX fraction). Each fraction was freeze-dried, and used for the following analyses .

A Tetrodotoxin-Like Substance

427

Thin-layer chromatography In the solvent system t-butanol - acetic acid - water, the GTX fraction showed three fluorescent spots, corresponding to GTX,, GTX,,, and GTX,, when visualized with 1 % H=O~ and 10% KOH and a pink spot of the same R, as TTX when visualized with Weber reagent (Fig. 2). In the pyridine - ethylacetate - acetic acid - water system, the GTX fraction showed a yellow spot of Rf 0 .42 when visualized with 1% HBO, and 10% KOH. This spot was not detected when 10% KOH was omitted, suggesting its being associated with TTX. The fraction exhibited two pink spots (R~ 0 .42 and 0.64) when visualized with Weber reagent. One of them coincided with TTX. It was found with preparative thin-layer chromatography that the lethal potency (as TTX) of the R~ 0 .42 spot accounted for about 0.7% of the total lethal potency. No attempts were made to estimate the lethality of the Rf 0.64 spot, since it overlapped with some GTXs. In either solvent system, the STX fraction gave two spots, one of which coincided with STX. The other spot seemed to be neoSTX from its R, value and green fluorescence . Electrophoresis As shown in Fig. 3, the GTX fraction gave six spots, four of which corresponded to GTX,-GTX,. One blue spot A (relative mobility to STX, O.SS) appeared between GTX, and GTX= and one yellow spot B (relative mobility to STX, 0.76) also appeared when visualized with 1 % H=O, and 10% KOH . Spot B was hardly detected when 10% KOH was omitted, suggesting that the spot is associated with TTX and not with paralytic shellfish poison components . Both these spots remain unidentified, but B might be the same as the

1.5 N AcUH (300rti )

Fia . 1 . Ax munox rteot~t.s of Atergatls floridus Tore FaoM w Bto-RHC 70 coLV~nv (0 .8 x 95 .5 cm) . APP~~B~ and ~P~ (95 ~ of the crabs were minced and extracted with 80bî acidified ethanol . The extract, defatted with chloroform, was subjected to an AmberGte IRC-SO column (2 .5 x 29 cm) . The toxic fraction eluted with IOb~ acetic acid was applied to a Bio-Gel P-2 column (6 .0 x SS cm). The toxic Fraction eluted with 0.1 N acetic acid was applied to the Bio-Rea 70 column . One mouse unit (Min is defined here as the amount of toxin required to kill a 20-g mouse in 1 S min .

42 8

T . NOGUCHI et al . Rf

Rf

0

000 O 000 00

~4z.~,t TTX GTXfi : STXfr

tert-butand-aceticacid-wafer (2 :1 :1) STX GTX3.z .~, TTX GTX1r. STXfr.

O

O

00 00

apt7 O ~O

pyrldk,e-ettylocebate-stalk acid-water (15 :5 :3 :4) FtG . 2 .

THIN-tarER cHxoMATOOxAPH,r of A . sPECi,~vs of TTX,

Jloridus Toxnvs, ToGETHEie wrrH AuTI~NTic STX wrro GTSs . The toxins were developed on a silica ge160 precoated plate (E . Merck), with a solvent system of tbutanol-acetic acid-water (2 :1 :1) (upper) and on a Whatman LHP-K precoated plate with a solvent system of pyridine - ethylacetate - acetic acid - water (15 :5 :3 :4) (lower) . Detection : 1 % H,O, and 104 KOH (left), and Weber reagent (right) .

R~ 0.42 spot . The STX fraction showed four spots, two of which were identified as STX and probably neoSTX (spot G) . The two less visible spots, D and E, remain unidentified . No spot whose relative mobility was the same as TTX appeared on electropherograms . Gas chromatography-mass spectrometry (GC-MS) In Fig. 4 is shown a mass chromatogram of the trimethylsilyl derivative induced from the GTX fraction, along with that of the trimethylsilyl derivative of the C9-base. All the mass fragment ion peaks of m/z 376, 392 and 407 appeared at a retention time of srx ~~,,,~

O+

TTX GTXfr. STXfr.

I I I I

I 0 1?YG . 3 .

ELECTROPHOAFSIS OF A,

0

I

0 I

05 Iielatlve moblllty

D

E I 1~0

f10l1difS TOXINS, ALONG WITH AUTHENTIC SPECIMENS OF TTX, STX AND GTXS . Electrophoresis was wnducted on S x 18 cm cellulose acetate membranes (Chemetron) in 0 .08 M Tris-HCI buffer (pH 8 .~ at 0 .8 mA/cm width for 60 min. Spots A-E, except C which was supposed to be neoSTX, remain unidentified . Spot B may be associated with TTX, since it was hardly detectable with 1 ß4 H,O, alone. Detection : 1 ~I H,O, and 10~ KOH .

A Tetrodotoxin-Like Substance

429

Ttne(min) 10 .i .. . .~ . . . .~ . ...~ .

n

15 .~ .

TIC 392 407 376 Tkne(min) 10 15 .~ .i . . . .~ . . . . . . . . .~ . . . .

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TIC 392 407 376

FIO . 4. MASS CHROMATOGRAMS OF THE TRA~TFIYLSILri DERIVATIVE FROM 2-AMWO-G HYDROXYME'rHYL-H-HYDROXYQUINAZOLINE (UPPHR) AND OF THE CORRHSPONDINO DERIVATIVE FROM TH8 wr Ywr ~-HYDROLYZED aTX FRACTION (LOWER). The column temperature was raised from 160°C to about 240°C at S°C/min . Mass

chromatograms were measured with a Hitachi aC-mass spectrometer M-80 at 70 eV . TIC = total ion carrent . 392, 407, 376 = masses of fragment ions, expressed in m/z .

13.9 min (trimethylsilyl derivative from the GTX fraction) and of 14.0 min (trimethylsilyl derivative from the C,-base) . The mass spectra of both peaks coincided well with each other (Fig . 5), indicating that GTX fraction contained a substance which gives rise to the C9-base when degraded with alkali, as does TTX (GOTO et al., 1%S).

430

T. NOGUCHI et al. 39z

407

50

318

203 200

300

('/.) 100

376 400 (mlz) 392

50

200

300

400 (mlz)

FIG . S . MASS SPECTRA OF THE TRIMETHYLSILYL DERIVATIVE FROM 2-AMINO-HYDROXYMECHYL-HHYDROXYQUINAZOLINE (UPPER) AND OF THE CORRESPONDING DERIVATIVE FROM THE ALKALIHYDROLYZED GTX FRACTION (LOR'ER) .

Mass spectra were measured with a Hitachi GC-mass spectrometer M-80 at 70 eV.

DISCUSSION

The results demonstrate that the A. floridus specimens contained a small amount of a substance possessing the TTX-specific quinazoline skeleton, in addition to STX and neoSTX as the major components and GTXs as a minor component. In thin-layer

A Tetrodotoxin-Like Substance

43 1

chromatography, the GTX fraction elicited a Weber-positive spot whose R~ coincided with TTX (Fig . 2) . However, no spot with the same mobility as TTX was detected in electrophoresis . Instead, two unidentified spots (A and B in Fig . 3) appeared . These results remind us of the TTX-associated toxin found in a small-sized gastropod mollusk, Zeuxis siquijorensis (NARITA et al., 1984) . The toxin of this gastropod was indistinguishable from TTX in thin-layer chromatography, but was clearly discriminated from TTX in electrophoresis (relative mobility of TTX, 0.7, vs that of TTX-associated substance, 0 .3) . Therefore, the quinazoline-possessing substance under consideration is supposed to be another TTX-associated or TTX-like compound . A. floridus specimens inhabiting Miura Peninsula, Kanagawa Prefecture, possess TTX as the major toxin, together with paralytic shellfish poisons as the minor (NOGUCHI et al., 1983). It follows, therefore, that the toxin composition of A . jloridus is qualitatively comparable between Ishigaki Island and Miura Peninsula, although markedly different quantitatively . In this connection, A . floridus crabs are highly resistant to both paralytic shellfish poisons and TTX (KOYAMA et al., 1983). On the other hand, we found that toxic pufferfish livers contain a nontoxic highmolecular weight substances) which releases TTX and some paralytic shellfish poisons on RNase digestion, and suggested that the high-molecular weight substance comes from some organisms) on which the pufferfish feeds (KODAMA et al., 1983x) . In fact, a trace amount of STX was detected in a highly toxic pufferfish liver (KODAMA et al., 1983b) . The present results, along with our recent finding with the A . floridus specimens (NocucHl et al., 1983), seem to favor the view that this crab is also made toxic by a food chains) involving some planktonic or other organisms . In this connection, KOTAKI et al. (1983) detected paralytic shellfish poisons in a red alga Jania sp. Because of its extremely low levels in the alga, however, its contribution to the toxicity of Atergatis crabs seems rather small .

Acknowledgements - The authors express their thanks to Director Dr A .

TOMORI and Mr . M Mutwcost-u, the Yaeyama Branch of the Okinawa Prefectural Fisheries Experimental Station, for their kind collaboration in collecting the crab specimens . This work was supported in part by a Grant-in-Aid for Scientific Research of the Ministry of Education, Science and Culture, Japan .

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KOYAMA, K ., Nocucxt, T ., Ueon, Y. and HASFItMOTO, K . (1981) Occurrence of neosaxitoxin and other paralytic shellfish poisons in toxic crabs belonging to the family Xanthidae . Bull. Jap. Soc. scient. Fish . 47, %5 . KOYAMA, K ., NocucHt, T ., Uzu, A. slid HASHIMOTO, K . (1983) Resistibility of toxic and nontoxic crabs against paralytic shellfish poison and tetrodotoxin . Bull. Jap. Soc. scient. Fish . 49, 485.

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et al.

NARITA, H., NOGUCHI, T., MARUYAMA, J., UEDA, Y., HASHIMOTO, K., WATANAHE, Y. 8nd HIDA, K. (1981) Occurrence of tetrodotoxin in a trumpet shell, "boshubora" Charonia sauliae. Bull. Jap. Soc. scient . Fish. 47, 935. NARITA, H., Nocucxl, T., MARUYAMA, J., NARA, M . and HASHIMOTO, K. (1984) Occurrence of a tetrodotoxinassociated substance in a gastropod, "hanaalushirogai" Zeuxis siquijorensis. Bull. Jap. Soc. scient. Fish. S0, 85 . Nooucxl, T., Kotvosu, S. and HASHIMOTO, Y. (1969) Identity of the crab toxin with saxitoxin . Toxicon 7, 325. Nooucxl, T., MARUYAMA, J., UEDA, Y., HASHIMOTO, K. and HARADA, T. (1981) Occurrence of tetrodotozin in the Japanese ivory shell Babylonia japonica. Bull. Jap. Soc . scient. Fish . 47, 909. Noaucxl, T., Uzu, A., KOYAMA, K., MARUYAMA, J., NAGASHIMA, Y. and HASHIMOTO, K. (1983) Occurrence of tetrodotoxin as the major toxin in a xanthid crab Atergatis floridus. Bull. Jap. Soc. scient. Fish . 49, 1887 . SUENAGA, K. and KoroKU, S. (1980) Detection of tetrodotoxin in autopsy material by gas chromatography . Arch . Tox. 44, 291 . USDA, Y., Nooucxl, T., OIVOUE, Y., KOYAMA, K., KONG, M. and HASHIMOTO, K. (1982) Occurrence of PSPinfested scallops in Ofunato Bay during 1976-1979 and investigation of responsible plankton . Bull. Jap. Soc. scient . Fish . 48, 455. YASUMOTO, T., OSHIMA, Y. and KONTA, T. (1981) Analysis of paralytic shellfish toxins of xanthid crabs in Okinawa. Bull. Jap. Soc. scient. Fish . 47, 957 .