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Heterogeneity of paralytic shellfish poisons. three new toxins from cultured Gony aulax tamarensis cells, Mya arenaria and Saxidomus giganteus
Comp Biochem. Physiol., 1977, Vol. 57C, pp. 31 to 34. Pergamon Press. Printed in Great Britain
HETEROGENEITY OF PARALYTIC SHELLFISH POISONS. THREE NEW TOXINS FROM C U L T U R E D G O N Y A U L A X T A M A R E N S I S CELLS, M Y A A R E N A R I A AND S A X I D O M U S G I G A N T E U S YASUKATSU OSH1MA, LAWRENCE J. BUCKLEY, MAKTOOB ALAM, AND YUZURU SHIMIZU Department of Pharmacognosy, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received 11 September 1976) Abstract--1. Three new toxins (gonyautoxin-IV, gonyautoxin-V, and neosaxitoxin) have been isolated
from cultured Gonyaulax tamarensis cells bringing the total number of paralytic shellfish poisons to seven. 2. Soft shell clams Mya arenaria, exposed to a bloom of G. tamarensis, have been found to contain gonyautoxin-IV and neosaxitoxin in different proportions to those found in the dinoflagellate. 3. Neosaxitoxin has been also isolated from Alaska butter clams, Saxidomus giganteus. This is the first report of the presence of a toxin other than saxitoxin in Alaska butter clams.
INTRODUCTION
MATERIALS AND METHODS
Organisms
Blooms of toxic dinoflagellates (red tides) have been reported in many parts of the world including the coasts of the U.S.A. and Canada. Certain species of Gonyaulax, particularly G. catenella in the North Pacific and G. tamarensis in the North Atlantic, produce potent neurotoxins which are concentrated by filter feeding organisms. Consumption of these exposed filter feeders produces paralytic shellfish poisoning (PSP), a severe and sometimes fatal form of food poisoning. Identification of the toxic principles involved is an urgent problem not only because of their great economic and public health significance, but also because of their particular mode of action. The paralytic shellfish toxins are of great pharmacological and biomedical interest since they belong to a small group of compounds which selectively blocks the passive influx of Na ÷ through excitable membranes (Narahashi, 1972; Narahashi et al., 1975). Saxitoxin, one of the most potent non-protein toxins known, has been isolated from California mussels (Mytilus californianus), Alaska butter clams (Saxidomus 9iganteus) and G. catenella by Schantz et al. (1957, 1966). Recent investigations of east coast PSP caused by blooms of G. tamarensis have resulted in the isolation of saxitoxin and three closely related toxins, gonyautoxin-I, -II and -III which were originally coded as GTX1, GTXz and GTX 3 (Ghazarossian et al., 1974; Shimizu et al., 1975b; Buckley et al., 1976). The structures of saxitoxin (Schantz et al., 1975a; Bordner et al., 1975) and gonyautoxin-II and -III (Shimizu et al., 1976) have recently been elucidated. In this communication we wish to report the isolation of three more new toxins from G. tamarensis cells, soft shell clams (Mya arenaria) and Alaska butter clams.
G. tamarensis cells used in this investigation were cultured in Guillard "F" medium according to the method reported previously (Shimizu et al., 1975a). When the cell numbers reached more than 6 x 103 c¢lls/ml, the organisms were harvested by continuous flow centrifugation.
Mya arenaria were collected in the fall of 1972 during a bloom of G. tamarensis along the north shore of Cape Ann, MA. The hepatopancreases were dissected and used for the extraction of toxins. Saxidomus giganteus were collected from Porpoise Island, AK, and the toxins were extracted from the siphons. Extractions and isolation of toxins The toxins were extracted with 80% ethanol adjusted to pH 2.0 with HC1 and isolated by column chromatography on Sephadex G-15 (Pharmacia Fine Chemicals), Bio-Gel P-2 and Bio-Rex 70 (BioRad Laboratories) according to the method reported by Shimizu et al., (1975b). Thin layer chromatography (TLC) TLC was performed on Silica Gel 60 precoated plates (E. Merck Laboratories) in pyridine, ethyl acetate, water and acetic acid (75:25:30:15 v/v). The spots were viewed under long wave u.v. light after spraying the plates with HzOz and heating (Buckley et al., 1976). Bioassay Toxicity was determined according to the method of United States Public Health Service originally described by Sommer & Meyer (1937), where one mouse unit (MU) is the amount of toxin necessary to kill a 20 g mouse in 15 min by the intraperitoneal injection. RESULTS
The toxins from G. tamarensis were isolated from 3101 of cell culture. As shown in Table 1, Bio-Rex 31
32
YASUKATSU
Table 1. Chromatographic behaviour of paralytic shellfish poisons Toxin
Elution order from Bio-Rex 70 column
TLC Ry*
GTX 5 GTX 4 GTX l GTX 2 GTX 3 neosaxitoxin saxitoxin
1 2 3 5 4 6 7
0.58 0.84 0.92 0.82 0.74 0.72 0.64
al.
et
OSHIMA,
16 rp°12
neoSTX
I
~]1.0
lilt
I
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.'"
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* Silica gel 60 plates developed in pyridine:ethyl acetate: acetic acid: water (75 :25 : 30: 15).
0 70 (H +) chromatography followed by TLC showed the presence of two new spots in the fractions eluted before the gonyautoxins isolated previously (Shimizu et al., 1975b). The two new toxins, gonyautoxins-IV and -V (coded as GTX4 and GTXs), were separated from the combined fractions in pure form by chromatography on a second Bio-Rex 70 (H +) column. Saxitoxin and a third new toxin (named neosaxitoxin) were isolated from the HC1 eluate of the first Bio-Rex 70 (H +) column. The separation of neosaxitoxin from saxitoxin was achieved by chromatography on a BioRex 70 (H +) column using an acetic acid gradient (0 to 1 M) (Fig. 1). The i.r. spectrum of neosaxitoxin, shown in Fig. 2, differed from those of both gonyautoxin-II and saxitoxin in that it showed a pronounced peak at 1770cm ;. The discovery of the three new toxins reported here brings the total number of toxins isolated from G. tamarensis to seven. These include five gonyautoxins (GTXI, GTX2, GTX3, GTX4 and GTXs) saxitoxin and neosaxitoxin. Gonyautoxin-II (about 25°.o) and neosaxitoxin (about 33Vo) made up the bulk of the poison in cells. Saxitoxin accounted for only about 5°/o of the toxicity. Soft shell clams (Mya arenaria) exposed to a bloom of G. tamarensis were found to contain four gonyautoxins, saxitoxin and neosaxitoxin, although in somewhat different proportions from that found in the dinoflagellate. GTX5 has not yet been detected in M. arenaria. The toxins in soft shell clams consisted of approximately 50~Jo gonyautoxin-II and 20~o saxitoxin. Only a trace of neosaxitoxin and GTX4 was found in the clams. Saxitoxin made up the majority of toxicity in Alaska butter clam siphons (90°Jo) with neosaxitoxin
. . . .
4000
i
. . . .
t
3000
. . . .
I
. . . .
I , t , I
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10 15 20 25 30 Fr. No.
Fig. 1. Separation of neosaxitoxin (neoSTX) and saxitoxin (STX) by a Bio-Rex 70 column (100 x 0.6 cm). Elution was carried out by means of a linear gradient of acetic acid from 0 to 1.0 M as indicated by dotted line. Fractions of 6 ml were collected. making up the remainder. None of the gonyautoxins were detected in Alaska butter clams. GTX¢ and GTX5 gave blue fluorescent spots on TLC plates after spraying with H202 and heating, similar to those described by Buckley et al. (1976) for the other gonyautoxins and saxitoxin. Treatment of neosaxitoxin under the same conditions gave a green fluorescent spot. Unlike saxitoxin and gonyautoxin-If (Shimizu et al., 1976), no fluorescent products could be isolated from a reaction mixture consisting of neosaxitoxin in 10/o H202 and 0.5 N N a O H , although a rapid loss of activity was observed. Heating neosaxitoxin in 1.0 N N a O H alone resulted in the production of two fluorescent degradation products which were separated by chromatography on Bio-Gel P-2. One product (u.v. 333 nm) showed blue fluorescence under long wave u.v. light, the other product (u.v. 370 nm) gave a yellow fluorescence. The chromatographic behaviour and toxicity of neosaxitoxin was unchanged after 24 hr in l N HC1. However, heating neosaxitoxin in a sealed ampule containing 7.5 N HC1 resulted in a 30~/o loss of activity and production of a product with a Rf value slight higher than that of neosaxitoxin. Reaction of saxitoxin under the same condition also resulted in a 30}~o loss of activity and yielded a product with an Ry value lower than that of saxitoxin. The prod-
,
i
1500
,
I
,
I
,
I
,
i
• 1000
i
,
i
J
I
,
I
i
I
i
I 500
•
,
, c m
Fig. 2. Infrared spectrum of neosaxitoxin dihydrochloride, taken with a Perkin-Elmer Model 521 in a KBr micropellet.
i_
Heterogeneity of paralytic shellfish poisons ucts obtained from the reaction of neosaxitoxin and saxitoxin in 7.5 N HC1 were clearly not identical. DISCUSSION
Large scale isolation of toxin from cultured G. tamarensis cells for structure determination and im-
proved detection methods for PSP led to the purification of three new toxins (GTX,, GTX5 and neosaxitoxin). In previous investigations we overlooked the presence of neosaxitoxin in G. tamarensis due to the very similar chromatographic behaviour of saxitoxin and neosaxitoxin. These two toxins had idential R I values in all TLC solvent systems tested except for pyridine:ethyl acetate:water:acetic acid (75:25:30:15 v/v) in which neosaxitoxin had a slightly higher R f value. Also neosaxitoxin and saxitoxin were both eluted from ion-exchange resin columns at nearly identical locations. The separation of neosaxitoxin from saxitoxin was effected only by careful high speed liquid chromatography on fine mesh Bio-Rex 70 (H+). Neosaxitoxin was clearly distinguished from saxitoxin by TLC and by production of several degradation products. The presence of neosaxitoxin in Alaska butter clams, although only a small percentage of the total toxicity, raises a serious question. In the past Alaska butter clams have been the major source of saxitoxin; however, the presence of a toxic component other than saxitoxin has never been recognized. Also, two saxitoxin standards in our possession were found not to contain neosaxitoxin. These observations raise two possibilities: (a) neosaxitoxin existed only in the particular clams we used, or (b) in the isolation procedure of Schantz et al. (1957) used in the past for saxitoxin, which involves removal of contaminants with sodium acetate buffer, neosaxitoxin was either transformed or destroyed and therefore not isolated. The possibility that neosaxitoxin is an artifact formed during our isolation process can be ruled out since the use of the same procedure on specimens containing only saxitoxin did not afford neosaxitoxin. Also, neosaxitoxin was unaffected by prolonged treatment with 1 N HC1 and treatment with 7.5 N HCI afforded a toxic product different from that obtained after the identical treatment of saxitoxin. In the case of saxitoxin, a similar experiment resulted in the isolation of decarbamyl saxitoxin (Ghazarossian et al., 1976). The presence of neosaxitoxin in Alaska butter clams raises one other interesting point. Although the origin of the toxin in Alaska butter clams has never been definitely established, the previously observed homogeneity of the toxin from Alaska butter clams, California sea mussels and G. catenella has been taken as evidence that the toxin originates in G. catenella by Schantz et al. (1975b). In contrast to earlier reports of the homogeneity of the toxin produced by G. catenella, six toxins including: GTX1, GTX:, GTXa, GTX4, GTX5 and saxitoxin have been isolated from shellfish and plankton samples collected during a red tide in Owase Bay, Japan (Oshima et al., 1976). Although Hashimoto et al. (1976) identified the causative organism as G. catenella on the basis of morphology, the toxins were identical to those found in G. tamarensis. The complexity and heterogeneity of the Gonyaulax toxins are also evident when one considers
t~ t). 57/IC -C
33
that some Gonyaulax sp. morphologically identical to G. tamarensis are nontoxic (Loeblich & Loeblich, 1975). Several significant differences were observed in the ratios of the different toxins in G. tamarensis cells and soft shell clams. No GTX s and only trace amounts of neosaxitoxin were found in soft shell clams which had a high saxitoxin content. G. tamarensis cells contained only trace amounts of saxitoxin and had a high neosaxitoxin content. Several explanations for the change in the relative concentrations of the toxins are possible, including preferential adsorption or metabolism in the clams and differences in the stability of the toxins during storage. All seven PSP toxins appear to have very similar chemical structures, possibly sharing a common nucleus. It has been shown that the only difference between saxitoxin and gonyautoxin-II and -III is the presence of an additional hydroxyl group at C-11 (Shimizu et al., 1976). GTX1, GTXz and GTX3 showed no qualitative difference in physiological activity although some quantitative differences were observed (T. Narahashi, personal communication). The production of similar blue fluorescent degradation products by reaction of the toxins with hydrogen peroxide also suggests a strong similarity in their structure. Only neosaxitoxin gave somewhat different degradation products. Neosaxitoxin appears to more closely resemble saxitoxin than the gonyautoxins because of its stronger basicity as suggested by its behaviour on Bio-Rex 70 (H +) columns, TLC and electrophoresis (to be published). Acknowledgements--This work was supported by HEW Grant Number FD00619 and the University of Rhode Island Sea Grant Program. The authors would like to thank Dr. E. J. Schantz, University of Wisconsin, for his generous gift of the saxitoxin standards, and Dr. Reichardt, University of Alaska, for the Saxidomus samples.
REFERENCES BORDNER J., THIESSEN W. E., BATES H. A. & RAPOPORT
H. (1975) The structure of a crystalline derivative of saxitoxin. The structure of saxitoxin. J. Am. Chem. Soc. 97, 6008-6012. BUCKLEY L. J., IKAWA M. & SASNER J. J. JR. (1976) Isolation of Gonyaulax tamarensis toxins from softshell clams
(Mya arenaria) and a thin layer chromatographic-fluorometric method for their detection. Affric. Food Chem.
24, 107 lll. GHAZAROSSIAN V. E., SCHANTZ E. J., SCHONOES H. K. &
STRONGF. M. (1974) Identification of a poison in toxic scallops from a Gonyaulax tamarensis red tide. Biochem. biophys. Res. Commun. 59. 1219-1225. GHAZAROSSIAN V. E., SCHANTZ E. J., SCHONOES H. K. d~
STRONGF. M. (1976) A biologically active acid hydrolysis product of saxitoxin. Biochem. biophys. Res. Commun. 68. 776-780. HASHIMOTOY., NOGUCH1T. & ADACm R. (1976) Occurrence of toxic bivalves in association with the bloom of Gonyaulax sp. in Owase Bay. Bull. Jap. Soc. Sci. Fish. 46, 671-676. LOEBLICHL. A. & LOEBLICHA. R. III (1975) The organism causing New England red tides: Gonyaulax excavata. Proceedings of the first international conference on toxic dinoflagellate blooms pp. 207 224. The Massachusetts Science and Technology Foundation, MA.
34
YASUKATSU OSHIMA, et al.
NARAHASHI T. (1972) Mechanism of action of tetrodotoxin and saxitoxin on excitable membranes. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1124-1132. NARAHASHI T., BRODWICK M. S. &, SCHANTZ E. J. (1975) Mechanism of action of a new toxin from Gonyaulax tamarensis. Environ. Lett. 9, 239-247. OSHIMA Y., FAELON W. E., SHIMIZU Y., NOGUCHI T. 8,= HASHIMOTO Y. (1976) Toxins of the Gonyaulax sp. and infested bivalves in Owase Bay. Bull. Jap. Soc. Sci. Fish. 42. 851 856. SCHANTZ E. J., MOLD J. D., STANGER D. W., SHANEL J., RIEL F. J., BOWDEN J. P., LYNCH J. M., WYLER R. S., RIEGEL B. 8,: SOMMERH. (1957) Paralytic shellfish poison. VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J. Am. Chem. Soc. 79, 5230-5235. SCHANTZ E. J., LYNCH J. M., VAYVADA G., MATSUMOTO K. &; RAPOPORT H. (1966) The purification and characterization of the poison produced by Gonyaulax catenella in axenic culture. Biochemistry 3, 1191-1195. SCHANTZ E. J., GHAZAROSS1AN V. E., SCHNOES H. K., STRONG F. M., SPRINGER J. P., PEZZANITE J. O. & CLARDY J. (1975a) The structure of saxitoxin. J. Am. Chem. Soc. 97, 1238-1239.
SCHANTZ E. J., GHAZAROSSIAN V. E., SCHNOES H. K., STRONG F. M., SPRINGER J. P., PEZZANITE J. O. CLARDY J. (1975b) Paralytic poisons from marine dinoflagellates. Proceedings of the first international conference on toxic dinoflagellate blooms, pp. 267 277, The Massachusetts Science and Technology Foundation, MA. SHIMIZU Y., ALAMM. tf,z FALLON W. E. (1975a) Purification and partial characterization of toxins from poisonous clams, Proceedings of the first international conference on toxic dinoflagellate blooms, pp. 275 285. The Massachusetts Science and Technology Foundation, MA. SHIMIZU Y., ALAM M., OSmMA Y. & FALLON W. E. (1975b) Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells. Biochem. biophys. Res. Commun. 66, 731 737. SHIMIZU Y., BUCKLEY L. J., ALAM M., OSHIMA Y., FALLON W. E., KASAI H., MIURA |., GULLO V. V. &; NAKANISHI K. (1976) Structures of Gonyautoxin II and III from the East Coast Toxic Dinoflagellate, Gonyaulax tamarensis, J. Am. Chem. Soc. 98, 5414-5416. SOMMER H. &- MEYER K. F. (1937) Paralytic shellfish poisoning. Arch. Pathol. 24, 560-598.
HETEROGENEITY OF PARALYTIC SHELLFISH POISONS. THREE NEW TOXINS FROM C U L T U R E D G O N Y A U L A X T A M A R E N S I S CELLS, M Y A A R E N A R I A AND S A X I D O M U S G I G A N T E U S YASUKATSU OSH1MA, LAWRENCE J. BUCKLEY, MAKTOOB ALAM, AND YUZURU SHIMIZU Department of Pharmacognosy, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received 11 September 1976) Abstract--1. Three new toxins (gonyautoxin-IV, gonyautoxin-V, and neosaxitoxin) have been isolated
from cultured Gonyaulax tamarensis cells bringing the total number of paralytic shellfish poisons to seven. 2. Soft shell clams Mya arenaria, exposed to a bloom of G. tamarensis, have been found to contain gonyautoxin-IV and neosaxitoxin in different proportions to those found in the dinoflagellate. 3. Neosaxitoxin has been also isolated from Alaska butter clams, Saxidomus giganteus. This is the first report of the presence of a toxin other than saxitoxin in Alaska butter clams.
INTRODUCTION
MATERIALS AND METHODS
Organisms
Blooms of toxic dinoflagellates (red tides) have been reported in many parts of the world including the coasts of the U.S.A. and Canada. Certain species of Gonyaulax, particularly G. catenella in the North Pacific and G. tamarensis in the North Atlantic, produce potent neurotoxins which are concentrated by filter feeding organisms. Consumption of these exposed filter feeders produces paralytic shellfish poisoning (PSP), a severe and sometimes fatal form of food poisoning. Identification of the toxic principles involved is an urgent problem not only because of their great economic and public health significance, but also because of their particular mode of action. The paralytic shellfish toxins are of great pharmacological and biomedical interest since they belong to a small group of compounds which selectively blocks the passive influx of Na ÷ through excitable membranes (Narahashi, 1972; Narahashi et al., 1975). Saxitoxin, one of the most potent non-protein toxins known, has been isolated from California mussels (Mytilus californianus), Alaska butter clams (Saxidomus 9iganteus) and G. catenella by Schantz et al. (1957, 1966). Recent investigations of east coast PSP caused by blooms of G. tamarensis have resulted in the isolation of saxitoxin and three closely related toxins, gonyautoxin-I, -II and -III which were originally coded as GTX1, GTXz and GTX 3 (Ghazarossian et al., 1974; Shimizu et al., 1975b; Buckley et al., 1976). The structures of saxitoxin (Schantz et al., 1975a; Bordner et al., 1975) and gonyautoxin-II and -III (Shimizu et al., 1976) have recently been elucidated. In this communication we wish to report the isolation of three more new toxins from G. tamarensis cells, soft shell clams (Mya arenaria) and Alaska butter clams.
G. tamarensis cells used in this investigation were cultured in Guillard "F" medium according to the method reported previously (Shimizu et al., 1975a). When the cell numbers reached more than 6 x 103 c¢lls/ml, the organisms were harvested by continuous flow centrifugation.
Mya arenaria were collected in the fall of 1972 during a bloom of G. tamarensis along the north shore of Cape Ann, MA. The hepatopancreases were dissected and used for the extraction of toxins. Saxidomus giganteus were collected from Porpoise Island, AK, and the toxins were extracted from the siphons. Extractions and isolation of toxins The toxins were extracted with 80% ethanol adjusted to pH 2.0 with HC1 and isolated by column chromatography on Sephadex G-15 (Pharmacia Fine Chemicals), Bio-Gel P-2 and Bio-Rex 70 (BioRad Laboratories) according to the method reported by Shimizu et al., (1975b). Thin layer chromatography (TLC) TLC was performed on Silica Gel 60 precoated plates (E. Merck Laboratories) in pyridine, ethyl acetate, water and acetic acid (75:25:30:15 v/v). The spots were viewed under long wave u.v. light after spraying the plates with HzOz and heating (Buckley et al., 1976). Bioassay Toxicity was determined according to the method of United States Public Health Service originally described by Sommer & Meyer (1937), where one mouse unit (MU) is the amount of toxin necessary to kill a 20 g mouse in 15 min by the intraperitoneal injection. RESULTS
The toxins from G. tamarensis were isolated from 3101 of cell culture. As shown in Table 1, Bio-Rex 31
32
YASUKATSU
Table 1. Chromatographic behaviour of paralytic shellfish poisons Toxin
Elution order from Bio-Rex 70 column
TLC Ry*
GTX 5 GTX 4 GTX l GTX 2 GTX 3 neosaxitoxin saxitoxin
1 2 3 5 4 6 7
0.58 0.84 0.92 0.82 0.74 0.72 0.64
al.
et
OSHIMA,
16 rp°12
neoSTX
I
~]1.0
lilt
I
I[ 8
o.s~ u
.'"
'X4Oo
"
o
~
* Silica gel 60 plates developed in pyridine:ethyl acetate: acetic acid: water (75 :25 : 30: 15).
0 70 (H +) chromatography followed by TLC showed the presence of two new spots in the fractions eluted before the gonyautoxins isolated previously (Shimizu et al., 1975b). The two new toxins, gonyautoxins-IV and -V (coded as GTX4 and GTXs), were separated from the combined fractions in pure form by chromatography on a second Bio-Rex 70 (H +) column. Saxitoxin and a third new toxin (named neosaxitoxin) were isolated from the HC1 eluate of the first Bio-Rex 70 (H +) column. The separation of neosaxitoxin from saxitoxin was achieved by chromatography on a BioRex 70 (H +) column using an acetic acid gradient (0 to 1 M) (Fig. 1). The i.r. spectrum of neosaxitoxin, shown in Fig. 2, differed from those of both gonyautoxin-II and saxitoxin in that it showed a pronounced peak at 1770cm ;. The discovery of the three new toxins reported here brings the total number of toxins isolated from G. tamarensis to seven. These include five gonyautoxins (GTXI, GTX2, GTX3, GTX4 and GTXs) saxitoxin and neosaxitoxin. Gonyautoxin-II (about 25°.o) and neosaxitoxin (about 33Vo) made up the bulk of the poison in cells. Saxitoxin accounted for only about 5°/o of the toxicity. Soft shell clams (Mya arenaria) exposed to a bloom of G. tamarensis were found to contain four gonyautoxins, saxitoxin and neosaxitoxin, although in somewhat different proportions from that found in the dinoflagellate. GTX5 has not yet been detected in M. arenaria. The toxins in soft shell clams consisted of approximately 50~Jo gonyautoxin-II and 20~o saxitoxin. Only a trace of neosaxitoxin and GTX4 was found in the clams. Saxitoxin made up the majority of toxicity in Alaska butter clam siphons (90°Jo) with neosaxitoxin
. . . .
4000
i
. . . .
t
3000
. . . .
I
. . . .
I , t , I
ZOO0
5
10 15 20 25 30 Fr. No.
Fig. 1. Separation of neosaxitoxin (neoSTX) and saxitoxin (STX) by a Bio-Rex 70 column (100 x 0.6 cm). Elution was carried out by means of a linear gradient of acetic acid from 0 to 1.0 M as indicated by dotted line. Fractions of 6 ml were collected. making up the remainder. None of the gonyautoxins were detected in Alaska butter clams. GTX¢ and GTX5 gave blue fluorescent spots on TLC plates after spraying with H202 and heating, similar to those described by Buckley et al. (1976) for the other gonyautoxins and saxitoxin. Treatment of neosaxitoxin under the same conditions gave a green fluorescent spot. Unlike saxitoxin and gonyautoxin-If (Shimizu et al., 1976), no fluorescent products could be isolated from a reaction mixture consisting of neosaxitoxin in 10/o H202 and 0.5 N N a O H , although a rapid loss of activity was observed. Heating neosaxitoxin in 1.0 N N a O H alone resulted in the production of two fluorescent degradation products which were separated by chromatography on Bio-Gel P-2. One product (u.v. 333 nm) showed blue fluorescence under long wave u.v. light, the other product (u.v. 370 nm) gave a yellow fluorescence. The chromatographic behaviour and toxicity of neosaxitoxin was unchanged after 24 hr in l N HC1. However, heating neosaxitoxin in a sealed ampule containing 7.5 N HC1 resulted in a 30~/o loss of activity and production of a product with a Rf value slight higher than that of neosaxitoxin. Reaction of saxitoxin under the same condition also resulted in a 30}~o loss of activity and yielded a product with an Ry value lower than that of saxitoxin. The prod-
,
i
1500
,
I
,
I
,
I
,
i
• 1000
i
,
i
J
I
,
I
i
I
i
I 500
•
,
, c m
Fig. 2. Infrared spectrum of neosaxitoxin dihydrochloride, taken with a Perkin-Elmer Model 521 in a KBr micropellet.
i_
Heterogeneity of paralytic shellfish poisons ucts obtained from the reaction of neosaxitoxin and saxitoxin in 7.5 N HC1 were clearly not identical. DISCUSSION
Large scale isolation of toxin from cultured G. tamarensis cells for structure determination and im-
proved detection methods for PSP led to the purification of three new toxins (GTX,, GTX5 and neosaxitoxin). In previous investigations we overlooked the presence of neosaxitoxin in G. tamarensis due to the very similar chromatographic behaviour of saxitoxin and neosaxitoxin. These two toxins had idential R I values in all TLC solvent systems tested except for pyridine:ethyl acetate:water:acetic acid (75:25:30:15 v/v) in which neosaxitoxin had a slightly higher R f value. Also neosaxitoxin and saxitoxin were both eluted from ion-exchange resin columns at nearly identical locations. The separation of neosaxitoxin from saxitoxin was effected only by careful high speed liquid chromatography on fine mesh Bio-Rex 70 (H+). Neosaxitoxin was clearly distinguished from saxitoxin by TLC and by production of several degradation products. The presence of neosaxitoxin in Alaska butter clams, although only a small percentage of the total toxicity, raises a serious question. In the past Alaska butter clams have been the major source of saxitoxin; however, the presence of a toxic component other than saxitoxin has never been recognized. Also, two saxitoxin standards in our possession were found not to contain neosaxitoxin. These observations raise two possibilities: (a) neosaxitoxin existed only in the particular clams we used, or (b) in the isolation procedure of Schantz et al. (1957) used in the past for saxitoxin, which involves removal of contaminants with sodium acetate buffer, neosaxitoxin was either transformed or destroyed and therefore not isolated. The possibility that neosaxitoxin is an artifact formed during our isolation process can be ruled out since the use of the same procedure on specimens containing only saxitoxin did not afford neosaxitoxin. Also, neosaxitoxin was unaffected by prolonged treatment with 1 N HC1 and treatment with 7.5 N HCI afforded a toxic product different from that obtained after the identical treatment of saxitoxin. In the case of saxitoxin, a similar experiment resulted in the isolation of decarbamyl saxitoxin (Ghazarossian et al., 1976). The presence of neosaxitoxin in Alaska butter clams raises one other interesting point. Although the origin of the toxin in Alaska butter clams has never been definitely established, the previously observed homogeneity of the toxin from Alaska butter clams, California sea mussels and G. catenella has been taken as evidence that the toxin originates in G. catenella by Schantz et al. (1975b). In contrast to earlier reports of the homogeneity of the toxin produced by G. catenella, six toxins including: GTX1, GTX:, GTXa, GTX4, GTX5 and saxitoxin have been isolated from shellfish and plankton samples collected during a red tide in Owase Bay, Japan (Oshima et al., 1976). Although Hashimoto et al. (1976) identified the causative organism as G. catenella on the basis of morphology, the toxins were identical to those found in G. tamarensis. The complexity and heterogeneity of the Gonyaulax toxins are also evident when one considers
t~ t). 57/IC -C
33
that some Gonyaulax sp. morphologically identical to G. tamarensis are nontoxic (Loeblich & Loeblich, 1975). Several significant differences were observed in the ratios of the different toxins in G. tamarensis cells and soft shell clams. No GTX s and only trace amounts of neosaxitoxin were found in soft shell clams which had a high saxitoxin content. G. tamarensis cells contained only trace amounts of saxitoxin and had a high neosaxitoxin content. Several explanations for the change in the relative concentrations of the toxins are possible, including preferential adsorption or metabolism in the clams and differences in the stability of the toxins during storage. All seven PSP toxins appear to have very similar chemical structures, possibly sharing a common nucleus. It has been shown that the only difference between saxitoxin and gonyautoxin-II and -III is the presence of an additional hydroxyl group at C-11 (Shimizu et al., 1976). GTX1, GTXz and GTX3 showed no qualitative difference in physiological activity although some quantitative differences were observed (T. Narahashi, personal communication). The production of similar blue fluorescent degradation products by reaction of the toxins with hydrogen peroxide also suggests a strong similarity in their structure. Only neosaxitoxin gave somewhat different degradation products. Neosaxitoxin appears to more closely resemble saxitoxin than the gonyautoxins because of its stronger basicity as suggested by its behaviour on Bio-Rex 70 (H +) columns, TLC and electrophoresis (to be published). Acknowledgements--This work was supported by HEW Grant Number FD00619 and the University of Rhode Island Sea Grant Program. The authors would like to thank Dr. E. J. Schantz, University of Wisconsin, for his generous gift of the saxitoxin standards, and Dr. Reichardt, University of Alaska, for the Saxidomus samples.
REFERENCES BORDNER J., THIESSEN W. E., BATES H. A. & RAPOPORT
H. (1975) The structure of a crystalline derivative of saxitoxin. The structure of saxitoxin. J. Am. Chem. Soc. 97, 6008-6012. BUCKLEY L. J., IKAWA M. & SASNER J. J. JR. (1976) Isolation of Gonyaulax tamarensis toxins from softshell clams
(Mya arenaria) and a thin layer chromatographic-fluorometric method for their detection. Affric. Food Chem.
24, 107 lll. GHAZAROSSIAN V. E., SCHANTZ E. J., SCHONOES H. K. &
STRONGF. M. (1974) Identification of a poison in toxic scallops from a Gonyaulax tamarensis red tide. Biochem. biophys. Res. Commun. 59. 1219-1225. GHAZAROSSIAN V. E., SCHANTZ E. J., SCHONOES H. K. d~
STRONGF. M. (1976) A biologically active acid hydrolysis product of saxitoxin. Biochem. biophys. Res. Commun. 68. 776-780. HASHIMOTOY., NOGUCH1T. & ADACm R. (1976) Occurrence of toxic bivalves in association with the bloom of Gonyaulax sp. in Owase Bay. Bull. Jap. Soc. Sci. Fish. 46, 671-676. LOEBLICHL. A. & LOEBLICHA. R. III (1975) The organism causing New England red tides: Gonyaulax excavata. Proceedings of the first international conference on toxic dinoflagellate blooms pp. 207 224. The Massachusetts Science and Technology Foundation, MA.
34
YASUKATSU OSHIMA, et al.
NARAHASHI T. (1972) Mechanism of action of tetrodotoxin and saxitoxin on excitable membranes. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1124-1132. NARAHASHI T., BRODWICK M. S. &, SCHANTZ E. J. (1975) Mechanism of action of a new toxin from Gonyaulax tamarensis. Environ. Lett. 9, 239-247. OSHIMA Y., FAELON W. E., SHIMIZU Y., NOGUCHI T. 8,= HASHIMOTO Y. (1976) Toxins of the Gonyaulax sp. and infested bivalves in Owase Bay. Bull. Jap. Soc. Sci. Fish. 42. 851 856. SCHANTZ E. J., MOLD J. D., STANGER D. W., SHANEL J., RIEL F. J., BOWDEN J. P., LYNCH J. M., WYLER R. S., RIEGEL B. 8,: SOMMERH. (1957) Paralytic shellfish poison. VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J. Am. Chem. Soc. 79, 5230-5235. SCHANTZ E. J., LYNCH J. M., VAYVADA G., MATSUMOTO K. &; RAPOPORT H. (1966) The purification and characterization of the poison produced by Gonyaulax catenella in axenic culture. Biochemistry 3, 1191-1195. SCHANTZ E. J., GHAZAROSS1AN V. E., SCHNOES H. K., STRONG F. M., SPRINGER J. P., PEZZANITE J. O. & CLARDY J. (1975a) The structure of saxitoxin. J. Am. Chem. Soc. 97, 1238-1239.
SCHANTZ E. J., GHAZAROSSIAN V. E., SCHNOES H. K., STRONG F. M., SPRINGER J. P., PEZZANITE J. O. CLARDY J. (1975b) Paralytic poisons from marine dinoflagellates. Proceedings of the first international conference on toxic dinoflagellate blooms, pp. 267 277, The Massachusetts Science and Technology Foundation, MA. SHIMIZU Y., ALAMM. tf,z FALLON W. E. (1975a) Purification and partial characterization of toxins from poisonous clams, Proceedings of the first international conference on toxic dinoflagellate blooms, pp. 275 285. The Massachusetts Science and Technology Foundation, MA. SHIMIZU Y., ALAM M., OSmMA Y. & FALLON W. E. (1975b) Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells. Biochem. biophys. Res. Commun. 66, 731 737. SHIMIZU Y., BUCKLEY L. J., ALAM M., OSHIMA Y., FALLON W. E., KASAI H., MIURA |., GULLO V. V. &; NAKANISHI K. (1976) Structures of Gonyautoxin II and III from the East Coast Toxic Dinoflagellate, Gonyaulax tamarensis, J. Am. Chem. Soc. 98, 5414-5416. SOMMER H. &- MEYER K. F. (1937) Paralytic shellfish poisoning. Arch. Pathol. 24, 560-598.