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Detection of tetrodotoxin-like compounds in two species of puffer fishes (Lagocephalus lunaris lunaris and Fugu niphobles)
Toxicron, Vol . 23, No. 2, pp. 331-336, 1983. Printed in Great Hrkain.
0041-0101/85 53 .00+ .00 ® 1985 PerBsmon Prag Ltd .
DETECTION OF TETRODOTOXIN-LIKE COMPOUNDS IN TWO SPECIES OF PUFFER FISHES (LAGOCEPHALUS LUNARIS L UNARIS AND FUGU NIPHOBLES) KAZUO SHIOMI, HAKARU INAOKA, HIDEAKI YAMANAKA and TAKEAKI KIKUCHI Laboratory of Food Hygienic Chemistry, Department of Food Science and Technology, Tokyo University of Fisheries, Tokyo 108, Japan (Accepted for publication 30 October 1984)
H. INAOKA, H. YnhInNnlu+ and T. Kltcucel . Detection of tetrodotoxin-like compounds in two species of puffer fishes (Lagotephalus lunaris lunaris and Fugu niphobles) . Taxiton 23, 331- 336, 1985 . - The toxins in the muscle, skin and liver of the puffer fish Lagocephalus lunaris lunaris and those in the skin and liver of another puffer fish Fugu niphobles were purified by successive column chromatography on activated charcoal, Bio-Gel P-2 and Bio-Rrx 70 and analyzed by electrophoresis and thin layer chromatography . Although, regardless of the tissues and species, tetrodotoxin was predominant and accounted for more than 90% of the total toxins, the following minor toxins were newly detected ; toxin A and toxin B in L . lunaris lunaris and toxin C in F. niphobles. Since these minor toxins, like tetrodotoxin, were positive to the Weber reagent and 10% KOH, but negative to 1 % H,O,, they were assumed to be tetrodotoxin-like compounds. Interestingly, the electrophoresic and thin layer chromatographic anâlyses showed that toxin A and toxin C are probably identical with AFT, and AFT,, the minor toxins in the crab Atergotis Jloridus from Chiba, Japan, respectively . K . SH1OM1,
INTRODUCTION
(TTX), a potent neurotoxin, is widely used in many laboratories as an important pharmacological reagent because of its ability to block selectively the sodium channels on the nerve membrane . It was first isolated from the Japanese puffer fish Fugu (Spheroides) rubripes (TSUDA and KAWAMURA, 1953 ; GOTO et al., 1965) and later from the following diverse animals : newts of the genus Taricha (BROWN and MOSHER, 1963; WAKELY et al., 1966); the goby Gobius criniger (NOGUCHI aril HASHIMOTO, 1973); Costa Rican frogs of the genus Atelopus (KIM et al., 1975); the blue-ringed octopus Hapalochlaena maculosa (SHEUMACK et al., 1978); three species of marine gastropods, Charonia sauliae (NARITA et al., 1981), Babylonia japonica (NOGUCHI et al., 1981 ; YASUMOTO et al., 1981a) and Tutufa lissostoma (NOGUCHI et al., 1984); the starfish Astropecten polyacanthus (NooucHl et al., 1982); the crab Atergatisfloridus (NOGUCHI et al., 1983) . Among those animals, the Costa Rican frog A . chiriquiensis (KIM et al., 1975), the octopus H. maculosa (SAVAGE and HOWDEN, 1977) and the gastropod B. japonica (YASUMOTO et al., 1981a) contain neurotoxins other than TTX. In addition, the presence of TTX-like compounds was reported in some gobies other than G. criniger (SLAM et al., 1977). As to puffer fish toxins, TTX has been believed to be the sole toxic compound since the first isolation from F. rubripes. However, the presence of toxins other than TTX was recently reported in the liver of Fugu (Takifugu) pardalis (KODAMA et al., 1983). In the course of purification of TTX from the muscle of puffer fish Lagocephalus lunaris TETRODOTOXIN
331
33 2
K. SHIOMI et al.
lunaris, we also detected two new toxins which behaved in thin layer chromatography and electrophoresis differently than TTX, which was present as the major toxin. This study was undertaken to confirm whether or not the above finding is true for other tissues of the same species and also for another species of puffer fish Fugu niphobles. MATERIALS AND METHODS Specimens of L. lunaris lunaris were caught in the Sea of East China in October 1981 and those of F. niphobles at Kominato, Chiba Prefecture, Japan, in June 1983 . All specimens were shipped to our laboratory
and kept at -20°C until use. Male mice (ddY strain) weighing about 20 g were purchased from Sankyo Labo Service, Tokyo, Japan. Activated charcoal was purchased from Kokusan Chemical Works, Tokyo, Japan; Bio-Gel P-2 and Bio-Rex 70 from Bio-Red Laboratories, Richmond, VA, U .S .A.; praoated silica gel plates from E. Merck, Darmstadt, West Germany; cellulose acetate strips from Sartorius, GSttingen, West Germany. TTX (citrate salt) was obtained from Sankyo Co ., Tokyo, Japan. Gonyautoxins 1-4 (GTX,~) were gifts of Dr Oshima, Faculty of Agriculture, Tohoku University, Japan. A mixture of AFT,_,, the toxins of the crab A. floridus from the Chiba Prefecture, Japan, was prepared as reported previously (StttoMr et al., 1982). Other chemical reagents were of analytical grade. Fxtraction acrd pur~cation of toxins Two female specimens of L. lunarls lunaris were separately used . In this paper these specimens were named
specimen 1 (body weight 9B0 g, total length 34 em) and specimen 2 (body weight 1400 g, total length 38 cm). The muscle, skin and liver were removed from each . In the case of F. niphobles, each sample of skin and liver was wmbined from 14 specimens (13 male specimens and 1 female specimen, body weight 63-113 g, total length 15 -18 cm) since this species was too small to investigate individually . Each tissue was extracted 3 times with S volumes of 75s1e aqueous ethanol containing l~h acetic acid. The extract was concentrated to remove ethanol and defatted 3 times with an equal volume of chloroform . The aqueous phase was chromatographed successively on columns of activated charcoal and Bio-Gel P-2 according to the method of Kornxc et al. (1981) . The partially purified toxin thus obtained was finally applied to a Hio-Rex 70 column (H' form, 1 x 100 cm). After washing with 250 ml of water, the column was eluted by a linear gradient method with 0-0.03 N acetic acid, S00 ml total volume, followed by 200 ml of 1 .5 N acetic acid . Thin layer chromatography (TLC) and electrophoresis
TLC on precoated silica gel plates and electrophoresis on cellulose acetate strips were performed as reported previously (SxtoMt et al., 1982). TTX, GTX,~ and AFT,_, were used as the reference toxins . After each run, the plate or strip was sprayed with 1% H,O, or l0~lo KOH and heated at 110°C for 1S min. Fluorescent spots were visualized under u.v . light (365 nm). Toxins were also detected by the Weber reagent (Wsaea, 1928). In order to check the lethality of the visualized compounds, each compound was isolated by preparative electrophoresis and subjected to mouse assay, by which the relative ratio in lethality of those compounds was computed . Mouse assay
Test solutions of 1 ml were injected i.p . into mice . In both cases, of TTX and TTX-like compounds, the lethality was determined by the dose-survival time relation of TTX (Knwwe~TA, 1978) and expressed in terms of mouse unit (MU) . One MU was defined as the amount of toxins required to kill a 20 g mouse in 30 min. RESULTS
The lethal potencies of the L. lunaris lunaris and F. niphobles specimens are summarized in Table 1 . As is generally known (HARADA, 1979), the high potency of muscle was observed with specimens 1 and 2 of L. lunaris lunaris. In contrast the muscle of F. niphobles was non-toxic (<5 MU/g tissue). The toxins from each tissue of both species were adsorbed by activated charcoal and Bio-Gel P-2 and could be eluted with acidic solvents . In Bio-Rex 70 column chromatography they were also adsorbed by the column and eluted quantitatively with 0-0.03 N acetic acid. The 1 .5 N acetic acid eluate contained no detectable amounts of toxins . As typical examples, the elution patterns from the Bio-Rex 70 column, together with their behavior in TLC and electrophoresis, are illustrated in Fig. 1 for the toxins
33 3
Toxins in Puffer Fishes
TAHLE
Species
Lagocephalus lunarls lunaris
1.
LETHAL POTENCY AND TOXIN COMPOSITION OF TWO SPECIES OF PUFFER FISHES Toxin composition (% of lethality}*
Specimen "
Tissue
Lethalityt (MU/g tissue)
1
Muscle Liver Muscle Skin Liver
121 140 96 261 116 708
93 92 97 99 100 99
Skin Liver
175 427
99 99
2 Fuge niphobles
Skia
TTX
Toxin A Toxin B
3 8 3 0 0 1
Toxin
C
4 0 0 1 0 0 1 1
"Two specimens were used separately in the case of L. lunaris lunaris, whileeach tissue was combined from 14 specimens in the case of F. niphobles. tpne MU was defined as the amount of toxin required to kill a 20 g mouse in 30 min. *Each toxin was isolated from the toxic fractions obtained in Bio-Rex 70 column chromatography by preparative electrophoresis on cellulose acetate strips with 0.08 M Tris - HCI buffer (pH 8.7). The relative ratio of each toxin to the total lethality was determined by mouse assay.
from the muscle of L. lunaris lunaris (specimen 1) and in Fig. 2 for those from the skin of When monitored by lethality to mice, a sharp toxin peak was observed in
F. niphobles.
,,,-0.03 0.02
0.01 .u u Q
i 50
GT X l-4 AFT 1-3
(b) 2 G OToxfnA
0 0 0 0 Taucin B 0
Fr . No .
100
cm 6
~a~ 03
1 . ELUTION PATTERN (a) FROM A BIO-REX 7O COLUMN OF Lagocephalus lunarls lunarls (SPECIMEN 1) AND ANALYSES F1ß.
.
u ro u
'~ 500 ô
Fr. 76 - 93 Fr. 94 , 95 Fr. 96-103 TTX
z
0
0
Rf 1.0
Tox~A o odfauc~ 9 0
°3 °t
THE TOXINS IN THE MUSCLE OF OF THE TOXIC FRACTIONS 6Y ELECTROPHORESIS (b) AND THIN LAYER CHROMATOGRAPHY (C). The toxins (17,000 MIn were applied to a Bio-Rex 70 column (H' form, 1 x 100 cm) equilibrated with water. After washing with 250 ml of water, the column was eluted by a linear gradient method wih 0-0.03 N acetic acid, 500 ml total volume . Fractions of 4 ml were collected at a flow rate of 33 ml/hr. Electrophoresis was carried out oa cellulose acetate stripa with 0.08 M Tris-HCI buffer (pH 8.7) at 0.4 mA/cm for 1 S min. Thin layer chromatography was performed oa precoated silica gel plates with a solvent system of pyridine - ethyl acetate- acetic acid -water (73 : 35 : 1 S : 30). The toxins were detected by the Weber reagent, 10"lo KOH or 1% H,O,. TTX, tetrodotoxln ; GTX,~,gonysutoxins 1-4; AFT,_,, Atergatis,/lorldus toxins 1-3.
K . SHIOMI et al.
334
OA3
1500 0.02
Ë w ~ 1000
v
'ir 500 .x
d Q 50 (b)
Fr. 82-91 Fr. 92 , 93 Fr. 94-97 TTX GTX 1-4 AFT 1-3 FIG .
i.
Z
2
Fr . No . cm
4_6~ ~ 0 0 0
100 (c) .5 0
Rf 1 10
~°°~,C~
01~ 03
ELUTION PATTERN (a) FROM A Bl0-REX 7O COLUMN OF THE TOXINS IN THE SKIN OF
Fugu
n1phObleS AND ANALYSES OF THE TOXIC FRACTIONS BY ELECTROPHORESIS (b) AND THIN LAYER CHROMATOGRAPHY (C) .
The toxins (10,300 MU) were chromatographed on Bio-Rex 70 . The experimental methods were as described in Fig . 1 .
both cases; it was attributed to TTX, judging from the results in TLC and electrophoresis. In the muscle of L . lunaris lunaris (Fig. 1), two new toxins (toxins A and B), which were distinguishable from TTX in TLC and electrophoresis, were detected . Both toxins, which were easily isolated by preparative electrophoresis, caused the same symptoms in mice as TTX and killed the mice within 30 min. As shown in Table 1, although their contents were very low as compared with that of TTX, at least one of the two toxins was detected in all the tissues of the two specimens, with the one exception of the skin of specimen 2, which contained only TTX. In the case of the skin of F. niphobles (Fig. 2), neither toxin A nor toxin B were found, but the presence of another new toxin (toxin C), which apparently differed from TTX, was indicated by TLC and electrophoresis . The symptoms in mice given toxin C were the same as those given TTX . Toxin C was also detected in the liver of the same species. As in the case of L . lunaris lunaris, TTX was much more predominant than toxin C, which accounted for only 1 % of the total toxins contained in both tissues of F. niphobles (Table 1). None of toxins A, B and C coincided with either one of GTX,~ in TLC and electrophoresis, but, surprisingly, toxin A and toxin C exhibited the same behaviors as AFT, and AFT, from the toxic crab A. floridus, respectively (Figs. 1 and 2). It was also noticeable that AFT, gave the same R~ value and mobility as TTX. The newly found toxins were all positive to the Weber reagent. When sprayed with 10% KOH and heated, these toxins were converted into fluorescent compounds which could be easily detected under u.v. light. However, they were practically negative to 1 % H,O,, since the reaction was much less sensitive than the KOH reaction . Accordingly, even when the fluorescent spots were detected, they were very vague. TTX and AFT,_, were also positive to the Weber reagent and 10% KOH and negative to 1 % H,Ol. Only GTX,~ gave positive reactions to all of the reagents used.
Toxins in Puffer Fishes
33 5
DISCUSSION
Although TTX was certainly the major toxin, as expected, the presence of three new toxins, distinguishable from TTX in chromatographic and electrophoresis behaviors, was established in the puffer fishes L. lunaris lunaris and F. niphobles. Toxins A and B were present in the former species and toxin C in the latter. Similarly, KODAMA et al. (1983) recently detected saxitoxin (STX) and an unknown toxin, which was supposed to be a TTX-like or GTX-like compound, in the liver of F. pardalis. Therefore, puffer fishes may possess toxins, probably as minor components, other than TTX in their tissues. Toxins A, B and C showed the same behaviors on activated charcoal and Bio-Gel P-2 as TTX and also gave the same reactions to the Weber reagent, 10% KOH and 1 % HBO, as TTX. Despite the detection of STX in F. pardalis by KODAMA et al. (1983), the three toxins were clearly distinguished from paralytic shellfish poisons in that they were negative to 1 % H=O=, while paralytic shellfish poisons such as the GTX, used in this study were positive. These results strongly suggest that the newly found toxins in the two species of puffer fishes are chemically related to TTX. As regards the toxins of A. floridus, it has previously been reported that STX and neoSTX are predominant in specimens from tropical or subtropical areas (YASUMOTO et al., 1981b; KOYAMA et al., 1981 ; RAJ et al., 1983). On the other hand, when we examined specimens from Chiba Prefecture, Japan, a temperate area, we found the greater part of toxins not in the STX fraction but in the GTX fraction ($HIOMI et al., 1982). Therefore, although the toxins were not identical with the known GTXs, they were supposed to be GTX-like toxins and were tentatively named AFT,_, . However, the major toxin in specimens from a temperate area was recently isolated and spectrometrically identified as TTX (NOGUCHI et al., 1983). The present study also revealed that, like TTX ,AFT,_, were positive to 10% KOH but negative to 1 % HBO, and that AFT, could not be distinguished from TTX in TLC and electrophoresis. It is, therefore, concluded that AFT l is none other than TTX and that AFT, and AFT, are not GTX-like but TTX-like toxins . The reason why specimens of A. floridus from tropical or subtropical areas possess STX and neoSTX, whereas those from temperate areas possess TTX and TTX-like toxins still remains unsolved. Acknowledgements - The authors are grateful to Dr M. Ctttaw, Research Station, Agriculture Canada, Vineland Station, Ontario, Canada, for revising this manuscript. This study was partly supported by a grant from the Ministry of Health and Welfare of Japan . REFERENCES BteowN, M . S. and MosxEx, H . S . (1%3) Tarichatoxin: isolation and purification. Science, N. Y. 140, 295 . Et.wM, K . S ., FUHRMAN, F . A ., KtM, Y . H . and Mosrtt:e, H . S . (1977) Neurotoxins from three species of California goby : Cleveiandia ios, Acanthogobius Jlavimanus and Güiichthys mirabilis. Taxiton 15, 45 . Goro, T ., Ktst-u, Y ., TwtcwtiwsHt, S. and Htawrw, Y . (1%S) Tetrodotoxin. Tetrahedron 21, 2059 . HARADA, Y. (1979) Classification and toxiwlogical examination of "fugu" imported from Fot~ttosa. J. Food Hyg. Soc. Japan 20, 437 . Kwwwawrw, T . (1978) Assay method for tetrodotoxin . In : Food Hygiene Examination Manual, Vol . 2, p. 232 (Environmental Health Huresu, Ministry of Health and Welfare of Japan) . Tokyo : Japan Food Hygiene Association . Ktnt, Y . H ., BROWN, G . B ., MOSHER, H . S . and Futtxn~wrt, F . A . (1975) Tetrodotoxin : occurrence in atelopid frogs of Costa Rica . Science, N. Y. 1>!9, 151 . KODAMw, M ., OGATA, T ., Nooucttt, T ., MARUYAMA, J . Snd HASHIMOTO, K . (1983) Occurrence of saxitoxin and other toxins in the liver of the pufferfish Tak(jugu pardalis. Toxirnn 21, 897 . Korwxt, Y ., OsHtntw, Y. and YASUMOTO, T . (1981) Analysis of paralytic shellfish toxins of marine snails . Bull. Jap. Soc. scient. Fish. 47, 943 .
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KOYAMA, K., Noaucrtt, T., Üt:Dw, Y. and Hwsxtmtoro, K. (1981) Occurrence of neosaxitoxin and otherparalytic shellfish poisons in toxic crabs belonging to the family Xanthidae. Bull. Jap. Soc. sciertt. Fish . 47, 965. Nwxtrw, H., NoaucHt, T., Mwaurwhtw, J., Ut:nw, Y ., HASHIMOTO, K., Wwrwtvwat=., Y. and HIDA, K. (1981) Oavrrence of tetrodotoxin in a trumpet shell, "boshubora" Charonia saullae. Bull. Jap. Soc. srtertt. Fish. 47, 933. Nocucttt, T. and HwsHtrforo, Y. (1973) Isolation of tetrodotoxin from a goby, Gobius crirtiger. Toxirnrt 11, 305. Noaucet, T., MARUYAMw, J., Li6DA, Y., HwsHtMOro, K. and Hww+ow, T. (1981) Oceurrence of tctrodotoxin in the Japanese ivory shell Babylonia japonica. Bull. Jap. Soc. scient. Fish. 47, 909. Noaucet, T., NARITA, H., MARUYAMA, J. SIId HASHIMOTO, K. (1982) Tetrodotoxin in a starfish Astropectert polyacanthus, in association with toxification of a trumpet shell, "boshubora" Charortia sauliae . Bull. Jap. Soc. sriertt. Fish . 48, 1173 . Noaucet, T., Uzu, A., KOYAMw, K., MwxuYwtww, J., Nwawswtww, Y. and Hwsttutoro, K. (1983) Occurrence of tctrodotoxin as the major toxin in a xanthid crab Atergatisjloridus . Bull. Jap. Soc. sciertt. Fish. 49, 1887 . Nooucet, T., MARUYAMA, J., NARI'rA, H. and HASIitMOTO, K. (1984) Occurrence of tetrodotoxin in the gastropod mollusk Tutyla ltssostoma (frog shell) . Toxirnrt 22, 219. Rw~, U., HwQ, H., OSHIMA, Y. eIId YASUMOTO, T. (1983) The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands. Toxicort 21, 347. Swvwct:, 1. V. E. and Hownsrt, M. E. H. (1977) Hapalotoxin, a second lethal toxin from the octopus Hapalochlaena maculosa. Taxirnn 15, 463 . Sttt7ut~twctc, D. D., Hownnrt, M . E. H., Spt?tvce, I. and Qunvrt, R. J. (1978) Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin . Science, N. Y. 199, 188. SxtoMt, K., INAOKw, H., YAMANAKA, H. and lütcucttt, T. (1982) Occurrence of a large amount of gonysutoxins in a xanthid crab Atergatisj]orldus from Chiba. Bull. Jap. Soc. scient. Fish. 48, 1407 . TSUDA, K. and Kwwwntuxw, M. (1933) The constituents of the ovaries of globefish-VIII . Studies on tetrodotoxin (1). CJrem. plurrm . Bull., Tokyo 1, 112. Wwttta.v, J. F., FUHRMAN, G. J., Futttttawn, F. A., Ftscttea, H . G. and MOSHER, H. S. (1966) The occurrence of tetrodotoxin (tarichatoxin) in Amphibia and the distribution of the toxin in the organs of newts (Taricha). Toxicort 3, 195. WEBER, C. J. (1928) The determination of the guanidine bases in urine. J. biol. Chem . 7g, 465. YASUMOTO, T., Ostttntw, Y., Hoswtu, M. and MtYAKOBIiI, H. (1981a) Occurrence of tetrodotoxin in the ivory shell Babylonia japonica from Wakasa Bay. Bull. Jap. Soc. sciertt. Fish . 47, 929. YASUMOTO, T., OSHIMA, Y. and Koxrw, T. (1981b) Analysis of paralytic shellfish toxins of xanthid crabs in Okinawa. Bull. Jap. Soc. sciertt. Fish. 47, 937.
0041-0101/85 53 .00+ .00 ® 1985 PerBsmon Prag Ltd .
DETECTION OF TETRODOTOXIN-LIKE COMPOUNDS IN TWO SPECIES OF PUFFER FISHES (LAGOCEPHALUS LUNARIS L UNARIS AND FUGU NIPHOBLES) KAZUO SHIOMI, HAKARU INAOKA, HIDEAKI YAMANAKA and TAKEAKI KIKUCHI Laboratory of Food Hygienic Chemistry, Department of Food Science and Technology, Tokyo University of Fisheries, Tokyo 108, Japan (Accepted for publication 30 October 1984)
H. INAOKA, H. YnhInNnlu+ and T. Kltcucel . Detection of tetrodotoxin-like compounds in two species of puffer fishes (Lagotephalus lunaris lunaris and Fugu niphobles) . Taxiton 23, 331- 336, 1985 . - The toxins in the muscle, skin and liver of the puffer fish Lagocephalus lunaris lunaris and those in the skin and liver of another puffer fish Fugu niphobles were purified by successive column chromatography on activated charcoal, Bio-Gel P-2 and Bio-Rrx 70 and analyzed by electrophoresis and thin layer chromatography . Although, regardless of the tissues and species, tetrodotoxin was predominant and accounted for more than 90% of the total toxins, the following minor toxins were newly detected ; toxin A and toxin B in L . lunaris lunaris and toxin C in F. niphobles. Since these minor toxins, like tetrodotoxin, were positive to the Weber reagent and 10% KOH, but negative to 1 % H,O,, they were assumed to be tetrodotoxin-like compounds. Interestingly, the electrophoresic and thin layer chromatographic anâlyses showed that toxin A and toxin C are probably identical with AFT, and AFT,, the minor toxins in the crab Atergotis Jloridus from Chiba, Japan, respectively . K . SH1OM1,
INTRODUCTION
(TTX), a potent neurotoxin, is widely used in many laboratories as an important pharmacological reagent because of its ability to block selectively the sodium channels on the nerve membrane . It was first isolated from the Japanese puffer fish Fugu (Spheroides) rubripes (TSUDA and KAWAMURA, 1953 ; GOTO et al., 1965) and later from the following diverse animals : newts of the genus Taricha (BROWN and MOSHER, 1963; WAKELY et al., 1966); the goby Gobius criniger (NOGUCHI aril HASHIMOTO, 1973); Costa Rican frogs of the genus Atelopus (KIM et al., 1975); the blue-ringed octopus Hapalochlaena maculosa (SHEUMACK et al., 1978); three species of marine gastropods, Charonia sauliae (NARITA et al., 1981), Babylonia japonica (NOGUCHI et al., 1981 ; YASUMOTO et al., 1981a) and Tutufa lissostoma (NOGUCHI et al., 1984); the starfish Astropecten polyacanthus (NooucHl et al., 1982); the crab Atergatisfloridus (NOGUCHI et al., 1983) . Among those animals, the Costa Rican frog A . chiriquiensis (KIM et al., 1975), the octopus H. maculosa (SAVAGE and HOWDEN, 1977) and the gastropod B. japonica (YASUMOTO et al., 1981a) contain neurotoxins other than TTX. In addition, the presence of TTX-like compounds was reported in some gobies other than G. criniger (SLAM et al., 1977). As to puffer fish toxins, TTX has been believed to be the sole toxic compound since the first isolation from F. rubripes. However, the presence of toxins other than TTX was recently reported in the liver of Fugu (Takifugu) pardalis (KODAMA et al., 1983). In the course of purification of TTX from the muscle of puffer fish Lagocephalus lunaris TETRODOTOXIN
331
33 2
K. SHIOMI et al.
lunaris, we also detected two new toxins which behaved in thin layer chromatography and electrophoresis differently than TTX, which was present as the major toxin. This study was undertaken to confirm whether or not the above finding is true for other tissues of the same species and also for another species of puffer fish Fugu niphobles. MATERIALS AND METHODS Specimens of L. lunaris lunaris were caught in the Sea of East China in October 1981 and those of F. niphobles at Kominato, Chiba Prefecture, Japan, in June 1983 . All specimens were shipped to our laboratory
and kept at -20°C until use. Male mice (ddY strain) weighing about 20 g were purchased from Sankyo Labo Service, Tokyo, Japan. Activated charcoal was purchased from Kokusan Chemical Works, Tokyo, Japan; Bio-Gel P-2 and Bio-Rex 70 from Bio-Red Laboratories, Richmond, VA, U .S .A.; praoated silica gel plates from E. Merck, Darmstadt, West Germany; cellulose acetate strips from Sartorius, GSttingen, West Germany. TTX (citrate salt) was obtained from Sankyo Co ., Tokyo, Japan. Gonyautoxins 1-4 (GTX,~) were gifts of Dr Oshima, Faculty of Agriculture, Tohoku University, Japan. A mixture of AFT,_,, the toxins of the crab A. floridus from the Chiba Prefecture, Japan, was prepared as reported previously (StttoMr et al., 1982). Other chemical reagents were of analytical grade. Fxtraction acrd pur~cation of toxins Two female specimens of L. lunarls lunaris were separately used . In this paper these specimens were named
specimen 1 (body weight 9B0 g, total length 34 em) and specimen 2 (body weight 1400 g, total length 38 cm). The muscle, skin and liver were removed from each . In the case of F. niphobles, each sample of skin and liver was wmbined from 14 specimens (13 male specimens and 1 female specimen, body weight 63-113 g, total length 15 -18 cm) since this species was too small to investigate individually . Each tissue was extracted 3 times with S volumes of 75s1e aqueous ethanol containing l~h acetic acid. The extract was concentrated to remove ethanol and defatted 3 times with an equal volume of chloroform . The aqueous phase was chromatographed successively on columns of activated charcoal and Bio-Gel P-2 according to the method of Kornxc et al. (1981) . The partially purified toxin thus obtained was finally applied to a Hio-Rex 70 column (H' form, 1 x 100 cm). After washing with 250 ml of water, the column was eluted by a linear gradient method with 0-0.03 N acetic acid, S00 ml total volume, followed by 200 ml of 1 .5 N acetic acid . Thin layer chromatography (TLC) and electrophoresis
TLC on precoated silica gel plates and electrophoresis on cellulose acetate strips were performed as reported previously (SxtoMt et al., 1982). TTX, GTX,~ and AFT,_, were used as the reference toxins . After each run, the plate or strip was sprayed with 1% H,O, or l0~lo KOH and heated at 110°C for 1S min. Fluorescent spots were visualized under u.v . light (365 nm). Toxins were also detected by the Weber reagent (Wsaea, 1928). In order to check the lethality of the visualized compounds, each compound was isolated by preparative electrophoresis and subjected to mouse assay, by which the relative ratio in lethality of those compounds was computed . Mouse assay
Test solutions of 1 ml were injected i.p . into mice . In both cases, of TTX and TTX-like compounds, the lethality was determined by the dose-survival time relation of TTX (Knwwe~TA, 1978) and expressed in terms of mouse unit (MU) . One MU was defined as the amount of toxins required to kill a 20 g mouse in 30 min. RESULTS
The lethal potencies of the L. lunaris lunaris and F. niphobles specimens are summarized in Table 1 . As is generally known (HARADA, 1979), the high potency of muscle was observed with specimens 1 and 2 of L. lunaris lunaris. In contrast the muscle of F. niphobles was non-toxic (<5 MU/g tissue). The toxins from each tissue of both species were adsorbed by activated charcoal and Bio-Gel P-2 and could be eluted with acidic solvents . In Bio-Rex 70 column chromatography they were also adsorbed by the column and eluted quantitatively with 0-0.03 N acetic acid. The 1 .5 N acetic acid eluate contained no detectable amounts of toxins . As typical examples, the elution patterns from the Bio-Rex 70 column, together with their behavior in TLC and electrophoresis, are illustrated in Fig. 1 for the toxins
33 3
Toxins in Puffer Fishes
TAHLE
Species
Lagocephalus lunarls lunaris
1.
LETHAL POTENCY AND TOXIN COMPOSITION OF TWO SPECIES OF PUFFER FISHES Toxin composition (% of lethality}*
Specimen "
Tissue
Lethalityt (MU/g tissue)
1
Muscle Liver Muscle Skin Liver
121 140 96 261 116 708
93 92 97 99 100 99
Skin Liver
175 427
99 99
2 Fuge niphobles
Skia
TTX
Toxin A Toxin B
3 8 3 0 0 1
Toxin
C
4 0 0 1 0 0 1 1
"Two specimens were used separately in the case of L. lunaris lunaris, whileeach tissue was combined from 14 specimens in the case of F. niphobles. tpne MU was defined as the amount of toxin required to kill a 20 g mouse in 30 min. *Each toxin was isolated from the toxic fractions obtained in Bio-Rex 70 column chromatography by preparative electrophoresis on cellulose acetate strips with 0.08 M Tris - HCI buffer (pH 8.7). The relative ratio of each toxin to the total lethality was determined by mouse assay.
from the muscle of L. lunaris lunaris (specimen 1) and in Fig. 2 for those from the skin of When monitored by lethality to mice, a sharp toxin peak was observed in
F. niphobles.
,,,-0.03 0.02
0.01 .u u Q
i 50
GT X l-4 AFT 1-3
(b) 2 G OToxfnA
0 0 0 0 Taucin B 0
Fr . No .
100
cm 6
~a~ 03
1 . ELUTION PATTERN (a) FROM A BIO-REX 7O COLUMN OF Lagocephalus lunarls lunarls (SPECIMEN 1) AND ANALYSES F1ß.
.
u ro u
'~ 500 ô
Fr. 76 - 93 Fr. 94 , 95 Fr. 96-103 TTX
z
0
0
Rf 1.0
Tox~A o odfauc~ 9 0
°3 °t
THE TOXINS IN THE MUSCLE OF OF THE TOXIC FRACTIONS 6Y ELECTROPHORESIS (b) AND THIN LAYER CHROMATOGRAPHY (C). The toxins (17,000 MIn were applied to a Bio-Rex 70 column (H' form, 1 x 100 cm) equilibrated with water. After washing with 250 ml of water, the column was eluted by a linear gradient method wih 0-0.03 N acetic acid, 500 ml total volume . Fractions of 4 ml were collected at a flow rate of 33 ml/hr. Electrophoresis was carried out oa cellulose acetate stripa with 0.08 M Tris-HCI buffer (pH 8.7) at 0.4 mA/cm for 1 S min. Thin layer chromatography was performed oa precoated silica gel plates with a solvent system of pyridine - ethyl acetate- acetic acid -water (73 : 35 : 1 S : 30). The toxins were detected by the Weber reagent, 10"lo KOH or 1% H,O,. TTX, tetrodotoxln ; GTX,~,gonysutoxins 1-4; AFT,_,, Atergatis,/lorldus toxins 1-3.
K . SHIOMI et al.
334
OA3
1500 0.02
Ë w ~ 1000
v
'ir 500 .x
d Q 50 (b)
Fr. 82-91 Fr. 92 , 93 Fr. 94-97 TTX GTX 1-4 AFT 1-3 FIG .
i.
Z
2
Fr . No . cm
4_6~ ~ 0 0 0
100 (c) .5 0
Rf 1 10
~°°~,C~
01~ 03
ELUTION PATTERN (a) FROM A Bl0-REX 7O COLUMN OF THE TOXINS IN THE SKIN OF
Fugu
n1phObleS AND ANALYSES OF THE TOXIC FRACTIONS BY ELECTROPHORESIS (b) AND THIN LAYER CHROMATOGRAPHY (C) .
The toxins (10,300 MU) were chromatographed on Bio-Rex 70 . The experimental methods were as described in Fig . 1 .
both cases; it was attributed to TTX, judging from the results in TLC and electrophoresis. In the muscle of L . lunaris lunaris (Fig. 1), two new toxins (toxins A and B), which were distinguishable from TTX in TLC and electrophoresis, were detected . Both toxins, which were easily isolated by preparative electrophoresis, caused the same symptoms in mice as TTX and killed the mice within 30 min. As shown in Table 1, although their contents were very low as compared with that of TTX, at least one of the two toxins was detected in all the tissues of the two specimens, with the one exception of the skin of specimen 2, which contained only TTX. In the case of the skin of F. niphobles (Fig. 2), neither toxin A nor toxin B were found, but the presence of another new toxin (toxin C), which apparently differed from TTX, was indicated by TLC and electrophoresis . The symptoms in mice given toxin C were the same as those given TTX . Toxin C was also detected in the liver of the same species. As in the case of L . lunaris lunaris, TTX was much more predominant than toxin C, which accounted for only 1 % of the total toxins contained in both tissues of F. niphobles (Table 1). None of toxins A, B and C coincided with either one of GTX,~ in TLC and electrophoresis, but, surprisingly, toxin A and toxin C exhibited the same behaviors as AFT, and AFT, from the toxic crab A. floridus, respectively (Figs. 1 and 2). It was also noticeable that AFT, gave the same R~ value and mobility as TTX. The newly found toxins were all positive to the Weber reagent. When sprayed with 10% KOH and heated, these toxins were converted into fluorescent compounds which could be easily detected under u.v. light. However, they were practically negative to 1 % H,O,, since the reaction was much less sensitive than the KOH reaction . Accordingly, even when the fluorescent spots were detected, they were very vague. TTX and AFT,_, were also positive to the Weber reagent and 10% KOH and negative to 1 % H,Ol. Only GTX,~ gave positive reactions to all of the reagents used.
Toxins in Puffer Fishes
33 5
DISCUSSION
Although TTX was certainly the major toxin, as expected, the presence of three new toxins, distinguishable from TTX in chromatographic and electrophoresis behaviors, was established in the puffer fishes L. lunaris lunaris and F. niphobles. Toxins A and B were present in the former species and toxin C in the latter. Similarly, KODAMA et al. (1983) recently detected saxitoxin (STX) and an unknown toxin, which was supposed to be a TTX-like or GTX-like compound, in the liver of F. pardalis. Therefore, puffer fishes may possess toxins, probably as minor components, other than TTX in their tissues. Toxins A, B and C showed the same behaviors on activated charcoal and Bio-Gel P-2 as TTX and also gave the same reactions to the Weber reagent, 10% KOH and 1 % HBO, as TTX. Despite the detection of STX in F. pardalis by KODAMA et al. (1983), the three toxins were clearly distinguished from paralytic shellfish poisons in that they were negative to 1 % H=O=, while paralytic shellfish poisons such as the GTX, used in this study were positive. These results strongly suggest that the newly found toxins in the two species of puffer fishes are chemically related to TTX. As regards the toxins of A. floridus, it has previously been reported that STX and neoSTX are predominant in specimens from tropical or subtropical areas (YASUMOTO et al., 1981b; KOYAMA et al., 1981 ; RAJ et al., 1983). On the other hand, when we examined specimens from Chiba Prefecture, Japan, a temperate area, we found the greater part of toxins not in the STX fraction but in the GTX fraction ($HIOMI et al., 1982). Therefore, although the toxins were not identical with the known GTXs, they were supposed to be GTX-like toxins and were tentatively named AFT,_, . However, the major toxin in specimens from a temperate area was recently isolated and spectrometrically identified as TTX (NOGUCHI et al., 1983). The present study also revealed that, like TTX ,AFT,_, were positive to 10% KOH but negative to 1 % HBO, and that AFT, could not be distinguished from TTX in TLC and electrophoresis. It is, therefore, concluded that AFT l is none other than TTX and that AFT, and AFT, are not GTX-like but TTX-like toxins . The reason why specimens of A. floridus from tropical or subtropical areas possess STX and neoSTX, whereas those from temperate areas possess TTX and TTX-like toxins still remains unsolved. Acknowledgements - The authors are grateful to Dr M. Ctttaw, Research Station, Agriculture Canada, Vineland Station, Ontario, Canada, for revising this manuscript. This study was partly supported by a grant from the Ministry of Health and Welfare of Japan . REFERENCES BteowN, M . S. and MosxEx, H . S . (1%3) Tarichatoxin: isolation and purification. Science, N. Y. 140, 295 . Et.wM, K . S ., FUHRMAN, F . A ., KtM, Y . H . and Mosrtt:e, H . S . (1977) Neurotoxins from three species of California goby : Cleveiandia ios, Acanthogobius Jlavimanus and Güiichthys mirabilis. Taxiton 15, 45 . Goro, T ., Ktst-u, Y ., TwtcwtiwsHt, S. and Htawrw, Y . (1%S) Tetrodotoxin. Tetrahedron 21, 2059 . HARADA, Y. (1979) Classification and toxiwlogical examination of "fugu" imported from Fot~ttosa. J. Food Hyg. Soc. Japan 20, 437 . Kwwwawrw, T . (1978) Assay method for tetrodotoxin . In : Food Hygiene Examination Manual, Vol . 2, p. 232 (Environmental Health Huresu, Ministry of Health and Welfare of Japan) . Tokyo : Japan Food Hygiene Association . Ktnt, Y . H ., BROWN, G . B ., MOSHER, H . S . and Futtxn~wrt, F . A . (1975) Tetrodotoxin : occurrence in atelopid frogs of Costa Rica . Science, N. Y. 1>!9, 151 . KODAMw, M ., OGATA, T ., Nooucttt, T ., MARUYAMA, J . Snd HASHIMOTO, K . (1983) Occurrence of saxitoxin and other toxins in the liver of the pufferfish Tak(jugu pardalis. Toxirnn 21, 897 . Korwxt, Y ., OsHtntw, Y. and YASUMOTO, T . (1981) Analysis of paralytic shellfish toxins of marine snails . Bull. Jap. Soc. scient. Fish. 47, 943 .
336
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KOYAMA, K., Noaucrtt, T., Üt:Dw, Y. and Hwsxtmtoro, K. (1981) Occurrence of neosaxitoxin and otherparalytic shellfish poisons in toxic crabs belonging to the family Xanthidae. Bull. Jap. Soc. sciertt. Fish . 47, 965. Nwxtrw, H., NoaucHt, T., Mwaurwhtw, J., Ut:nw, Y ., HASHIMOTO, K., Wwrwtvwat=., Y. and HIDA, K. (1981) Oavrrence of tetrodotoxin in a trumpet shell, "boshubora" Charonia saullae. Bull. Jap. Soc. srtertt. Fish. 47, 933. Nocucttt, T. and HwsHtrforo, Y. (1973) Isolation of tetrodotoxin from a goby, Gobius crirtiger. Toxirnrt 11, 305. Noaucet, T., MARUYAMw, J., Li6DA, Y., HwsHtMOro, K. and Hww+ow, T. (1981) Oceurrence of tctrodotoxin in the Japanese ivory shell Babylonia japonica. Bull. Jap. Soc. scient. Fish. 47, 909. Noaucet, T., NARITA, H., MARUYAMA, J. SIId HASHIMOTO, K. (1982) Tetrodotoxin in a starfish Astropectert polyacanthus, in association with toxification of a trumpet shell, "boshubora" Charortia sauliae . Bull. Jap. Soc. sriertt. Fish . 48, 1173 . Noaucet, T., Uzu, A., KOYAMw, K., MwxuYwtww, J., Nwawswtww, Y. and Hwsttutoro, K. (1983) Occurrence of tctrodotoxin as the major toxin in a xanthid crab Atergatisjloridus . Bull. Jap. Soc. sciertt. Fish. 49, 1887 . Nooucet, T., MARUYAMA, J., NARI'rA, H. and HASIitMOTO, K. (1984) Occurrence of tetrodotoxin in the gastropod mollusk Tutyla ltssostoma (frog shell) . Toxirnrt 22, 219. Rw~, U., HwQ, H., OSHIMA, Y. eIId YASUMOTO, T. (1983) The occurrence of paralytic shellfish toxins in two species of xanthid crab from Suva barrier reef, Fiji Islands. Toxicort 21, 347. Swvwct:, 1. V. E. and Hownsrt, M. E. H. (1977) Hapalotoxin, a second lethal toxin from the octopus Hapalochlaena maculosa. Taxirnn 15, 463 . Sttt7ut~twctc, D. D., Hownnrt, M . E. H., Spt?tvce, I. and Qunvrt, R. J. (1978) Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin . Science, N. Y. 199, 188. SxtoMt, K., INAOKw, H., YAMANAKA, H. and lütcucttt, T. (1982) Occurrence of a large amount of gonysutoxins in a xanthid crab Atergatisj]orldus from Chiba. Bull. Jap. Soc. scient. Fish. 48, 1407 . TSUDA, K. and Kwwwntuxw, M. (1933) The constituents of the ovaries of globefish-VIII . Studies on tetrodotoxin (1). CJrem. plurrm . Bull., Tokyo 1, 112. Wwttta.v, J. F., FUHRMAN, G. J., Futttttawn, F. A., Ftscttea, H . G. and MOSHER, H. S. (1966) The occurrence of tetrodotoxin (tarichatoxin) in Amphibia and the distribution of the toxin in the organs of newts (Taricha). Toxicort 3, 195. WEBER, C. J. (1928) The determination of the guanidine bases in urine. J. biol. Chem . 7g, 465. YASUMOTO, T., Ostttntw, Y., Hoswtu, M. and MtYAKOBIiI, H. (1981a) Occurrence of tetrodotoxin in the ivory shell Babylonia japonica from Wakasa Bay. Bull. Jap. Soc. sciertt. Fish . 47, 929. YASUMOTO, T., OSHIMA, Y. and Koxrw, T. (1981b) Analysis of paralytic shellfish toxins of xanthid crabs in Okinawa. Bull. Jap. Soc. sciertt. Fish. 47, 937.