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A comparison of the biological effects of paralytic shellfish poisons from clam, mussel and dinoflagellate
Toxlcon, 1971, Vol . 9, pp. 139-144 . Per~amon Press. Printed in Great Britain
A COMPARISON OF THE BIOLOGICAL EFFECTS OF PARALYTIC SHELLFISH POISONS FROM CLAM, MUSSEL AND DINOFLAGELLATE M. H. EvAxs A.R.C. Institute of Animal Physiology, Babraham, Cambridge, England (Accepted for pttbliaotlole 13 drrgv.,
Abebract~xitoxin, the paralytic shellfish poison from the clam Saxidomus gigartteus, has been tested on mice, frog sciatic nerves, filaments from the rat cauda equine and the frog sartorius muscle . The effects on all of these preparations were indistinguishable from the effects of paralytic shellfish poisons purified from the mussel Mytilus calljornianus and the plankton dinofiagellate Gonyaulax cateneRa. The results of the experiments support the hypothesis that the poisons from all three sources are identical. INTRODUCTION
Solono>~R, Meyer and their associates assembled evidence that the paralytic shellfish poison found in California sea mussels (Mytilus californianus) originates in the plankton dinoflagellate Gonyaulax catenella (see So1rn~R and MExex, 1937 ; So~¢R et al., 1937). The circumstantial evidence that the paralytic shellfish poisons from these two sources are identical has received strong support from the chemical studies of Schantz and his colleagues, who have also produced evidence indicating that the paralytic shellfish poison from the Alaskan butter clam (Saxidorrtus giganteus) may have a similar identity (MOLD et al., 1957 ; ScxANZZ et al., 1957 ; Sci-r~rrrz et al., 1961 ; ScxexTZ et al., 1966). These workers have amassed a considerable collection of data on the chemical and physical properties of paralytic shellfish poisons extracted from the clam, mussel and dinoflagellate mentioned above, and these properties appear to be identical, within the limits of experimental error. Nevertheless, to exclude a possibility of a qualitative error, it was thought that further confirmation of identity in physiological actions should be sought in a series of tests on various biological preparations . The present paper describes the results of these tests, which have not revealed any differences in the effects of the three poisons. The work was done at the suggestion of Dr. E. J. Schantz, to whom I am grateful for the supply of the poisons. MATERIALS AND METHODS
The paralytic shellfish poisons from S. giganteus, M. californianus and G. catenella were supplied by Dr. E. J. Schantz in the form of powders accurately weighed in small sealed ampoules . For use in the present experiments the entire content of each ampoule was dissolved in 0~1 N HCl to make a stock solution having a concentration of 100 wg per ml. It was thought advisable to avoid the use of ethanol (recommended by ScxArrrz et al., 1958, to inhibit growth of moulds) ; therefore the solutions were made under aseptic conditions and sealed in small sterilized ampoules, each holding a few ml of the solution, 139
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which were kept refrigerated. Stored under these conditions there was no evidence of any deterioration of the material. Further dilutions were made, as required, from the contents of these stock ampoules . The mouse Ln~ for each of the three poisons was determined by intraperitoneal (i.p.) administration . The assay procedures conformed to the general principles recommended by SCHANTZ et al. (1958) and by McFnitRSx (1959), except that the following modification was introduced : because some loss by leakage is sometimes seen when the poison is given in 1 ml, the volume administered was reduced to 050 ml in the case of mice of 20 g body weight. When the weight of the mouse differed from 20 g the volume injected was varied proportionately, so as to maintain the dose constant in terms of ng poison per g mouse. The mean weight ofthe mice used was 238 g ; there were no significant differences between the mean weights of the three assay lots. Each of the three poisons was assayed on a 24 mouse lot, divided into four groups of six. The poisons were given in four doses, diluted with distilled water so that the doses formed a geometrical progression, chosen to produce less than 50 ~ mortality in two groups and 50 ~ mortality or more in the other two groups . This allowed the results to be analysed with WEtL'S (1952) tables . The following preparations were used in comparing the effects of the three poisons : compound action potentials conducted along partially desheathed frog sciatic nerve and along filaments from the rat cauda equina ; resting membrane potentials and evoked endplate potentials of frog sartorius muscle . Only the middle region of the frog nerve was desheathed . This region was in the central compartment of a 3~hambered nerve bath and imgated with Ringer's solution flowing at 2-3 ml/min . The poisons were tested by adding them to this solution to give the appropriate concentrations . The two ends of the nerve were not desheathed and were in oil in the end compartments of the nerve bath for stimulation and recording of the conducted compound action potential . The rat cauda equina filaments were treated similarly except that they did not need to be desheathed, having negligible permeability bamers . The Ringer's solutions were made up to the following composition . Frog Ringer : NaCI, 110 ; KCI, 2~5 ; CaCla, 1~8 ; Glucose, 5~6 ; Tris HCl buffer pH 7~4, 3 mM/1. For rat nerve : NaCI, 150 ; KCI, 5~6 ; CaClB, 2~2 ; Glucose, 5~6; Tris HCl buffer pH 7~4, 5 mM/l. In the experiments on end-plate potentials the muscle was paralysed by the addition to the Ringer's solution of D-tubocurarine chloride (Tubarine miscible, Burroughs Wellcome & Co.) 1 ~~3 mg per 1., or MgC19 8-13 mM per 1. replacing an osmotically equivalent concentration of NaCI . The full details of all the experimental procedures used in these tests have been described in earlier publications (EV~xs, 1964, 1965, 1968, 1969a,b). RESULTS
LD~p Determination The analysis of the results of i.p. injection into mice indicated that there were no significant differences between the potencies of the clam, mussel and dinoflagellate poisons . The mouse Ln bu doses, calculated with the aid of Wßll.'S (1952) tables, and expressed as ng per 20 g body weight, were : S. giganteus : 200 ng (95 per cent confidence limits : 190-210) ; M. californianus: 211 ng (limits : 196-226); G. catenella: 2075 ng (limits : 193-222).
Paralytic Shellfish Poisons
141
In those mice that received more than a lethal dose, the behaviour of the animals before death was similar for all three poisons, and so also was the time elapsing between injection of the poison and death of the animal . Effects vn nerve conduction The poisons from all three sources caused a failure of conduction tivhen applied in vitro partially desheathed frog sciatic nerves, or to filaments from the rat cauda equina . to The compound action potential, recorded at one end of a frog sciatic nerve, was reduced to 50 per cent amplitude after 4-5 min application of any of the three poisons to the central desheathed part of the nerve in a concentration of 5 Fig per 1. The poisons were also tested at 10 leg per 1. and it can be seen from Fig. 1 that all three produced mV 4r
Ordinate : peak voltage of the group A compound action potentials (the zero level of each suoceasive graph is displaced downwards by 1 mV to avoid overlapping) . Abscissa : time in 2 min intervals. The black bar signals application of poison, l0 ~+.g per 1 ., to central desheathed part of nerve . Applications were repeated at intervals of 1 hr, the nerve being washed in between tests . ~ G7am saxitoxin ; O Mussel poison ; p Plankton poison .
quantitatively similar effects on the conducted action potentials . Each poison was applied for 6 min, after which the nerve was washed with Ringer's solution for 54 min before the next test. The nerve recovered completely during this washing period, so allowing six successive comparisons to be made on the same piece of nerve. The baseline of each successive graph is displaced downwards by 1 mV in Fig. 1 to avoid overlapping of the curves . It can be seen that the depressions produced by each of the poisons (applied during the time marked by the black bar) and the subsequent recoveries during the washing period are very similar. Figure 2 shows that similar results were obtained when the poisons were tested on the rat cauda equina . In this preparation there is usually a slow deterioration in the amplitude of the compound action potential, which makes quantitative assays less reliable. Nevertheless, the depressions and recoveries shown, to each of six successive exposures to the poisons at 5 wg per 1. for 10 min, resembled each other closely. The poisons were also
142
M. H. EVANS
FIG. 2. EFFSGT ON RAT CAUDA EQUINA NERVS. Symbols and co-ordinates as in Fig. 1. The black bar signals 10 min application of poison, 5 leg per l ., followed by 80 min wash .
tested at concentrations of 4 and 6 hg per 1. on other rat cauda equine preparations, again with results that were indistinguishable from each other. Effects on the resting potential across the cell membrane of the muscle fibre
Penetration of frog sartorius muscle fibres, with KCI-filled micropipettes, allowed the resting membrane potential to be recorded . The mean potential, from 24 experiments, was -885 mV (S.D . :6~9 mV). None of the poisons, when applied in concentrations of 8 or 10 wg per 1., produced any detectable change in the resting muscle membrane potential. Drift of amplifier baseline and electrode potentials sometimes amounted to a 2 mV change in the zero reference during a time similar to the duration of test, therefore any effect smaller than this drift could have escaped detection. The horizontal trace above the graphs of Fig. 3 is a potentiometric record of an experiment in which the muscle was exposed to clam poison, 10 tag per 1., during the 2 min period marked by the black bar . The membrane potential remained within the range -955 to -96 mV during the 8 min of the recording. Effects on the enaiplate potential
Intracellular recording of the end-plate potentials (e.p.p.) at the neuromuscularjtmction of the frog can be used to distinguish saxitoxin from tetrodotoxin (Evwrrs, 19696) and from the toxin of Gonyaulax tamarensis (Evens, to be published).
143
Paralytic She119sh Poisons
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EPFEC,T ON RE317NG MEMBRANE POTENTIAL AND EVOKED END-PLATE POTENTIAL OF FROG SARTORIUS MUSCLB .
The three graphs show the depression of e.p .p . caused by plankton poison (p), mussel poison (p) and clam saxitoxin ( ~) . The poisons were applied for 2 min, as shown by the black bar, in concentrations of 10 I"g per 1. The horizontal trace at the top of the figure is the membrane potential, recorded simultaneously during the exposure to the clam saxitoxin. The other two poisons showed a similar lack of effect on the resting membrane potential. The preparation was washed for 38 min between the tests. The muscle was paralysed by inclusion of D-tubocurarine 2 mg per 1. in all the test and washing Ringer's solutions.
Sartorius muscles were exposed to clam, mussel or dinoflagellate poison in concentrations of 8 or 10 Fig per I. while recordings of the e.p.p. were being made. In most of the experiments the three poisons gave identical results. In a few experiments some minor variations were recorded, but were too small to be considered significant. Figure 3 shows three superimposed graphs which were taken from tests on a frog sartorius muscle . The muscle was curarized ; all the solutions contained D-tubocurarine 2 mg per 1. In each run the muscle was exposed to one of the poisons (10 Fig per 1.) for 2 min followedby 38 min washingwith Ringer's solution which also contained n-tubocurarine 2 mg per 1. This allowed full recovery before the next test . The three graphs were all plotted from responses at the same end-plate ; no significant differences can be seen. All three poisons produced the same slow decline in the amplitude of the e.p.p. and the responses recovered steadily when the poison was washed out. Small variations in the speed of recovery are attributable to irregularities in the flow rate of the irrigating Ringer's solution. A few experiments were performed on muscles that had been paralysed with MgCI $ instead of curare . Application of the poisons to these preparations sometimes led to abrupt changes in e.p.p. amplitude, usually when the poison was being washed out after causing the usual fall in e.p.p. amplitude. Thes e abrupt changes are thought to be due to the motor axon becoming blocked by the poison in the presence of MgCI, (EvANS, 19696) . Those end-plates that showed an abrupt change of e.p.p. amplitude to one poison also showed similar abrupt changes when tested with either of the other two poisons. DISCUSSION
Comparison of the physical and chemical properties of the paralytic shellfish poisons from Saxidomus giganteus, Mytilus californiamis and Gonyaulaz catenella (summarized by ScxANTZ, 196 made it seem probable that all three sources yielded one and the same poisonous substance. Similarly, the poisons from the clam and the mussel appeared to
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M. H. EVANS
have identical actions when tested on the respiration, blood pressure and muscular reflexes in anaesthetized mammals (MuxTxA, 1960 ; KAO and NISHIYAMA, 1965). The results of further tests on the biological effects of the poisons from these three sources, reported in the present work, have not given any indication of differences in their actions. About 1964 the paralytic shellfish poison from Saxidomus giganteus was named saxitoxin (RAPOPORT et al., 1964 ; NISHIYAMA and KAO, 1964). Since then there has been a growing tendency to extend this name to include the paralytic shellfish poison from mussel and dinoflagellate . Some workers, including the present author, have been uneasy about this wider use of the name saxitoxin when evidence for the identity of the three toxins was not conclusive . Absolute proof of identity is still lacking, but the biological evidence presented in this paper is in full agreement with current views, based upon physical and chemical data, that clam saxitoxin and paralytic shellfish poison from M. californiums and from G. catenella are probably identical. Acknowledgements-I am indebted to Dr. E. J. Scxnxrz for the three samples of paralytic shellfish poison, and to Miss JEAxn: Tuu.ocH for her assistance . Some equipment was bought through U.S .P.H .S . Grant No . 5 ROS TW 00355.
REFERENCES Evntvs, M. H. (1964) Paralytic effects of `Paralytic Shellfish Poison' on frog nerve and muscle . Br. J. Pharrrmc . 22, 478. Evens, M. H. (1965) Cause of death in experimental paralytic shellfish poisoning (psp) . Br. J. exp. Path . 46, 245. Evens, M. H. (1968) The cauda equine of the rat : a convenient `desheathed' mammalian nerve preparation . J. Physiol. 194, S1P. EVAN3, M. H. (1969a). The effects of saxitoxin and tetrodotoxin on nerve conduction in the presence of lithium ions and of magnesium ions . Br. J. Pharmac. 36, 418. Eveivs, M. H. (1969b) Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction . Br . J. Pharmac. 36, 426. ICeo, C. Y. and NtstnveMe, A. (1965) Actions of saxitoxin on peripheral neuromuscular systems. J. Physiol. 180, 50 . McF.+RxEtv, E. F. (1959) Report on collaborative studies of the bioassay for paralytic shellfish poison . J. Ass. off agric. Chem. 42, 263. Moi.n, J. D., Bown~, J. P., Srerrcsx, D. W., Mevxsa, J. E., LYNCH, J. M., WYLEa, R. S., Scxerrrz, E. J . and Rmo~, B. (1957) Paralytic shellfish poison-VII . Evidence for the purity of the poison isolated from toxic clams and mussels. J. Am. them . Soc. 79, 5235 . MunTHe, E. F. (1960) Pharmacological study of poisons from shellfish and puffer fish . Ann. N.Y. Aced . Sci. 90, 820. NisFnveMe, A. and Iüo, C. Y. (1964) Presynaptic actions of saxitoxin at neuromuscular junction . Fedn Prat . Fedn Am . Sots exp. Biol. T3, 298. RAPOPORT, H., Bxowx, M. S., Ops~reßrarr, R. and SCHUeTT, W. (1964) Saxitoxin. Abstr. Pap. Am . them. Soc. (147th meeting), p. 3N. ScxeN~rz, E. J. (1967) Biochemical studies on purified Gonyaulax caterulla poison. In : Animal Toxins, p. 91 (RvssEr t,, F. E. and Seurroeas, P. R., Eds). Oxford : Pergamon Preas. Scxerrrz, E. J., Mot.n, J. D., Howean, W. L., BOWDEN, J. P., $TANGER, D. W., LnvcH, J. M., Wnv~r~s7e~Ete, O. P., DuTCH~re, J. D., Werxaas, D. R. and R~cet, B. (1961) Paralytic shellfish poison-VIiI . Some chemical and physical properties of purified clam and mussel poisons. Con. J. Chem . 39, 2117 . ScaexTZ, E. J., Mot h, J. D., STexasa, D. W., SHev~,, J., Rn;t., F. J., BOWDHN, J. P., LYxcH, J. M., Wvtea, R. S., R~L, B. and Sot~a, H. (1957) Paralytic shellfish poison-VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J. Am . them . Soc. 79, 5230. $CHANTZ, E. J., McFeweaN, E. F., Scxe~ß, M. L. and LEWis, K. H. (1958) Purified shellfish poison for bioassay standardization. J. Ass. off. agric. Chem . 41, 160. $CHANTZ, E. J., LYNCH, J. M., VeYVADA, G., MwzsuMO~ro, K. and RAPOPORT, H. (1966) The purification and characterization of the poison produced by Gonyaulax catenella in axenic culture. Biochemistry 5,1191 . Sort, H. and MBY~x, K. F. (1937) Paralytic shellfish poisoning. A.M.A . Arch . Path . 24, 560. Sonsngx, H., WHeooN, W. F., KOPOro, C. A. and Sro~t, R. (1937) Relation of paralytic shellfish poison to certain plankton organisms of the genus Gonyaulax. A.M.A . Arch . Path . 24, 537 . Wsn., C. S. (1952) Tables for convenient calculation of mediaa-effective dose (c.n,~ or sn~,) and instructions in their use. Biometrics S, 249.
A COMPARISON OF THE BIOLOGICAL EFFECTS OF PARALYTIC SHELLFISH POISONS FROM CLAM, MUSSEL AND DINOFLAGELLATE M. H. EvAxs A.R.C. Institute of Animal Physiology, Babraham, Cambridge, England (Accepted for pttbliaotlole 13 drrgv.,
Abebract~xitoxin, the paralytic shellfish poison from the clam Saxidomus gigartteus, has been tested on mice, frog sciatic nerves, filaments from the rat cauda equine and the frog sartorius muscle . The effects on all of these preparations were indistinguishable from the effects of paralytic shellfish poisons purified from the mussel Mytilus calljornianus and the plankton dinofiagellate Gonyaulax cateneRa. The results of the experiments support the hypothesis that the poisons from all three sources are identical. INTRODUCTION
Solono>~R, Meyer and their associates assembled evidence that the paralytic shellfish poison found in California sea mussels (Mytilus californianus) originates in the plankton dinoflagellate Gonyaulax catenella (see So1rn~R and MExex, 1937 ; So~¢R et al., 1937). The circumstantial evidence that the paralytic shellfish poisons from these two sources are identical has received strong support from the chemical studies of Schantz and his colleagues, who have also produced evidence indicating that the paralytic shellfish poison from the Alaskan butter clam (Saxidorrtus giganteus) may have a similar identity (MOLD et al., 1957 ; ScxANZZ et al., 1957 ; Sci-r~rrrz et al., 1961 ; ScxexTZ et al., 1966). These workers have amassed a considerable collection of data on the chemical and physical properties of paralytic shellfish poisons extracted from the clam, mussel and dinoflagellate mentioned above, and these properties appear to be identical, within the limits of experimental error. Nevertheless, to exclude a possibility of a qualitative error, it was thought that further confirmation of identity in physiological actions should be sought in a series of tests on various biological preparations . The present paper describes the results of these tests, which have not revealed any differences in the effects of the three poisons. The work was done at the suggestion of Dr. E. J. Schantz, to whom I am grateful for the supply of the poisons. MATERIALS AND METHODS
The paralytic shellfish poisons from S. giganteus, M. californianus and G. catenella were supplied by Dr. E. J. Schantz in the form of powders accurately weighed in small sealed ampoules . For use in the present experiments the entire content of each ampoule was dissolved in 0~1 N HCl to make a stock solution having a concentration of 100 wg per ml. It was thought advisable to avoid the use of ethanol (recommended by ScxArrrz et al., 1958, to inhibit growth of moulds) ; therefore the solutions were made under aseptic conditions and sealed in small sterilized ampoules, each holding a few ml of the solution, 139
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M . H. EVANS
which were kept refrigerated. Stored under these conditions there was no evidence of any deterioration of the material. Further dilutions were made, as required, from the contents of these stock ampoules . The mouse Ln~ for each of the three poisons was determined by intraperitoneal (i.p.) administration . The assay procedures conformed to the general principles recommended by SCHANTZ et al. (1958) and by McFnitRSx (1959), except that the following modification was introduced : because some loss by leakage is sometimes seen when the poison is given in 1 ml, the volume administered was reduced to 050 ml in the case of mice of 20 g body weight. When the weight of the mouse differed from 20 g the volume injected was varied proportionately, so as to maintain the dose constant in terms of ng poison per g mouse. The mean weight ofthe mice used was 238 g ; there were no significant differences between the mean weights of the three assay lots. Each of the three poisons was assayed on a 24 mouse lot, divided into four groups of six. The poisons were given in four doses, diluted with distilled water so that the doses formed a geometrical progression, chosen to produce less than 50 ~ mortality in two groups and 50 ~ mortality or more in the other two groups . This allowed the results to be analysed with WEtL'S (1952) tables . The following preparations were used in comparing the effects of the three poisons : compound action potentials conducted along partially desheathed frog sciatic nerve and along filaments from the rat cauda equina ; resting membrane potentials and evoked endplate potentials of frog sartorius muscle . Only the middle region of the frog nerve was desheathed . This region was in the central compartment of a 3~hambered nerve bath and imgated with Ringer's solution flowing at 2-3 ml/min . The poisons were tested by adding them to this solution to give the appropriate concentrations . The two ends of the nerve were not desheathed and were in oil in the end compartments of the nerve bath for stimulation and recording of the conducted compound action potential . The rat cauda equina filaments were treated similarly except that they did not need to be desheathed, having negligible permeability bamers . The Ringer's solutions were made up to the following composition . Frog Ringer : NaCI, 110 ; KCI, 2~5 ; CaCla, 1~8 ; Glucose, 5~6 ; Tris HCl buffer pH 7~4, 3 mM/1. For rat nerve : NaCI, 150 ; KCI, 5~6 ; CaClB, 2~2 ; Glucose, 5~6; Tris HCl buffer pH 7~4, 5 mM/l. In the experiments on end-plate potentials the muscle was paralysed by the addition to the Ringer's solution of D-tubocurarine chloride (Tubarine miscible, Burroughs Wellcome & Co.) 1 ~~3 mg per 1., or MgC19 8-13 mM per 1. replacing an osmotically equivalent concentration of NaCI . The full details of all the experimental procedures used in these tests have been described in earlier publications (EV~xs, 1964, 1965, 1968, 1969a,b). RESULTS
LD~p Determination The analysis of the results of i.p. injection into mice indicated that there were no significant differences between the potencies of the clam, mussel and dinoflagellate poisons . The mouse Ln bu doses, calculated with the aid of Wßll.'S (1952) tables, and expressed as ng per 20 g body weight, were : S. giganteus : 200 ng (95 per cent confidence limits : 190-210) ; M. californianus: 211 ng (limits : 196-226); G. catenella: 2075 ng (limits : 193-222).
Paralytic Shellfish Poisons
141
In those mice that received more than a lethal dose, the behaviour of the animals before death was similar for all three poisons, and so also was the time elapsing between injection of the poison and death of the animal . Effects vn nerve conduction The poisons from all three sources caused a failure of conduction tivhen applied in vitro partially desheathed frog sciatic nerves, or to filaments from the rat cauda equina . to The compound action potential, recorded at one end of a frog sciatic nerve, was reduced to 50 per cent amplitude after 4-5 min application of any of the three poisons to the central desheathed part of the nerve in a concentration of 5 Fig per 1. The poisons were also tested at 10 leg per 1. and it can be seen from Fig. 1 that all three produced mV 4r
Ordinate : peak voltage of the group A compound action potentials (the zero level of each suoceasive graph is displaced downwards by 1 mV to avoid overlapping) . Abscissa : time in 2 min intervals. The black bar signals application of poison, l0 ~+.g per 1 ., to central desheathed part of nerve . Applications were repeated at intervals of 1 hr, the nerve being washed in between tests . ~ G7am saxitoxin ; O Mussel poison ; p Plankton poison .
quantitatively similar effects on the conducted action potentials . Each poison was applied for 6 min, after which the nerve was washed with Ringer's solution for 54 min before the next test. The nerve recovered completely during this washing period, so allowing six successive comparisons to be made on the same piece of nerve. The baseline of each successive graph is displaced downwards by 1 mV in Fig. 1 to avoid overlapping of the curves . It can be seen that the depressions produced by each of the poisons (applied during the time marked by the black bar) and the subsequent recoveries during the washing period are very similar. Figure 2 shows that similar results were obtained when the poisons were tested on the rat cauda equina . In this preparation there is usually a slow deterioration in the amplitude of the compound action potential, which makes quantitative assays less reliable. Nevertheless, the depressions and recoveries shown, to each of six successive exposures to the poisons at 5 wg per 1. for 10 min, resembled each other closely. The poisons were also
142
M. H. EVANS
FIG. 2. EFFSGT ON RAT CAUDA EQUINA NERVS. Symbols and co-ordinates as in Fig. 1. The black bar signals 10 min application of poison, 5 leg per l ., followed by 80 min wash .
tested at concentrations of 4 and 6 hg per 1. on other rat cauda equine preparations, again with results that were indistinguishable from each other. Effects on the resting potential across the cell membrane of the muscle fibre
Penetration of frog sartorius muscle fibres, with KCI-filled micropipettes, allowed the resting membrane potential to be recorded . The mean potential, from 24 experiments, was -885 mV (S.D . :6~9 mV). None of the poisons, when applied in concentrations of 8 or 10 wg per 1., produced any detectable change in the resting muscle membrane potential. Drift of amplifier baseline and electrode potentials sometimes amounted to a 2 mV change in the zero reference during a time similar to the duration of test, therefore any effect smaller than this drift could have escaped detection. The horizontal trace above the graphs of Fig. 3 is a potentiometric record of an experiment in which the muscle was exposed to clam poison, 10 tag per 1., during the 2 min period marked by the black bar . The membrane potential remained within the range -955 to -96 mV during the 8 min of the recording. Effects on the enaiplate potential
Intracellular recording of the end-plate potentials (e.p.p.) at the neuromuscularjtmction of the frog can be used to distinguish saxitoxin from tetrodotoxin (Evwrrs, 19696) and from the toxin of Gonyaulax tamarensis (Evens, to be published).
143
Paralytic She119sh Poisons
too +to
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to
mv
s
.:~" ,, "":""
;":~ ::;';:;h :" ,s.' : '"Y
": ..
4 " 0
.
°°°
°°°°°
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FYO. 3.
EPFEC,T ON RE317NG MEMBRANE POTENTIAL AND EVOKED END-PLATE POTENTIAL OF FROG SARTORIUS MUSCLB .
The three graphs show the depression of e.p .p . caused by plankton poison (p), mussel poison (p) and clam saxitoxin ( ~) . The poisons were applied for 2 min, as shown by the black bar, in concentrations of 10 I"g per 1. The horizontal trace at the top of the figure is the membrane potential, recorded simultaneously during the exposure to the clam saxitoxin. The other two poisons showed a similar lack of effect on the resting membrane potential. The preparation was washed for 38 min between the tests. The muscle was paralysed by inclusion of D-tubocurarine 2 mg per 1. in all the test and washing Ringer's solutions.
Sartorius muscles were exposed to clam, mussel or dinoflagellate poison in concentrations of 8 or 10 Fig per I. while recordings of the e.p.p. were being made. In most of the experiments the three poisons gave identical results. In a few experiments some minor variations were recorded, but were too small to be considered significant. Figure 3 shows three superimposed graphs which were taken from tests on a frog sartorius muscle . The muscle was curarized ; all the solutions contained D-tubocurarine 2 mg per 1. In each run the muscle was exposed to one of the poisons (10 Fig per 1.) for 2 min followedby 38 min washingwith Ringer's solution which also contained n-tubocurarine 2 mg per 1. This allowed full recovery before the next test . The three graphs were all plotted from responses at the same end-plate ; no significant differences can be seen. All three poisons produced the same slow decline in the amplitude of the e.p.p. and the responses recovered steadily when the poison was washed out. Small variations in the speed of recovery are attributable to irregularities in the flow rate of the irrigating Ringer's solution. A few experiments were performed on muscles that had been paralysed with MgCI $ instead of curare . Application of the poisons to these preparations sometimes led to abrupt changes in e.p.p. amplitude, usually when the poison was being washed out after causing the usual fall in e.p.p. amplitude. Thes e abrupt changes are thought to be due to the motor axon becoming blocked by the poison in the presence of MgCI, (EvANS, 19696) . Those end-plates that showed an abrupt change of e.p.p. amplitude to one poison also showed similar abrupt changes when tested with either of the other two poisons. DISCUSSION
Comparison of the physical and chemical properties of the paralytic shellfish poisons from Saxidomus giganteus, Mytilus californiamis and Gonyaulaz catenella (summarized by ScxANTZ, 196 made it seem probable that all three sources yielded one and the same poisonous substance. Similarly, the poisons from the clam and the mussel appeared to
l44
M. H. EVANS
have identical actions when tested on the respiration, blood pressure and muscular reflexes in anaesthetized mammals (MuxTxA, 1960 ; KAO and NISHIYAMA, 1965). The results of further tests on the biological effects of the poisons from these three sources, reported in the present work, have not given any indication of differences in their actions. About 1964 the paralytic shellfish poison from Saxidomus giganteus was named saxitoxin (RAPOPORT et al., 1964 ; NISHIYAMA and KAO, 1964). Since then there has been a growing tendency to extend this name to include the paralytic shellfish poison from mussel and dinoflagellate . Some workers, including the present author, have been uneasy about this wider use of the name saxitoxin when evidence for the identity of the three toxins was not conclusive . Absolute proof of identity is still lacking, but the biological evidence presented in this paper is in full agreement with current views, based upon physical and chemical data, that clam saxitoxin and paralytic shellfish poison from M. californiums and from G. catenella are probably identical. Acknowledgements-I am indebted to Dr. E. J. Scxnxrz for the three samples of paralytic shellfish poison, and to Miss JEAxn: Tuu.ocH for her assistance . Some equipment was bought through U.S .P.H .S . Grant No . 5 ROS TW 00355.
REFERENCES Evntvs, M. H. (1964) Paralytic effects of `Paralytic Shellfish Poison' on frog nerve and muscle . Br. J. Pharrrmc . 22, 478. Evens, M. H. (1965) Cause of death in experimental paralytic shellfish poisoning (psp) . Br. J. exp. Path . 46, 245. Evens, M. H. (1968) The cauda equine of the rat : a convenient `desheathed' mammalian nerve preparation . J. Physiol. 194, S1P. EVAN3, M. H. (1969a). The effects of saxitoxin and tetrodotoxin on nerve conduction in the presence of lithium ions and of magnesium ions . Br. J. Pharmac. 36, 418. Eveivs, M. H. (1969b) Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction . Br . J. Pharmac. 36, 426. ICeo, C. Y. and NtstnveMe, A. (1965) Actions of saxitoxin on peripheral neuromuscular systems. J. Physiol. 180, 50 . McF.+RxEtv, E. F. (1959) Report on collaborative studies of the bioassay for paralytic shellfish poison . J. Ass. off agric. Chem. 42, 263. Moi.n, J. D., Bown~, J. P., Srerrcsx, D. W., Mevxsa, J. E., LYNCH, J. M., WYLEa, R. S., Scxerrrz, E. J . and Rmo~, B. (1957) Paralytic shellfish poison-VII . Evidence for the purity of the poison isolated from toxic clams and mussels. J. Am. them . Soc. 79, 5235 . MunTHe, E. F. (1960) Pharmacological study of poisons from shellfish and puffer fish . Ann. N.Y. Aced . Sci. 90, 820. NisFnveMe, A. and Iüo, C. Y. (1964) Presynaptic actions of saxitoxin at neuromuscular junction . Fedn Prat . Fedn Am . Sots exp. Biol. T3, 298. RAPOPORT, H., Bxowx, M. S., Ops~reßrarr, R. and SCHUeTT, W. (1964) Saxitoxin. Abstr. Pap. Am . them. Soc. (147th meeting), p. 3N. ScxeN~rz, E. J. (1967) Biochemical studies on purified Gonyaulax caterulla poison. In : Animal Toxins, p. 91 (RvssEr t,, F. E. and Seurroeas, P. R., Eds). Oxford : Pergamon Preas. Scxerrrz, E. J., Mot.n, J. D., Howean, W. L., BOWDEN, J. P., $TANGER, D. W., LnvcH, J. M., Wnv~r~s7e~Ete, O. P., DuTCH~re, J. D., Werxaas, D. R. and R~cet, B. (1961) Paralytic shellfish poison-VIiI . Some chemical and physical properties of purified clam and mussel poisons. Con. J. Chem . 39, 2117 . ScaexTZ, E. J., Mot h, J. D., STexasa, D. W., SHev~,, J., Rn;t., F. J., BOWDHN, J. P., LYxcH, J. M., Wvtea, R. S., R~L, B. and Sot~a, H. (1957) Paralytic shellfish poison-VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J. Am . them . Soc. 79, 5230. $CHANTZ, E. J., McFeweaN, E. F., Scxe~ß, M. L. and LEWis, K. H. (1958) Purified shellfish poison for bioassay standardization. J. Ass. off. agric. Chem . 41, 160. $CHANTZ, E. J., LYNCH, J. M., VeYVADA, G., MwzsuMO~ro, K. and RAPOPORT, H. (1966) The purification and characterization of the poison produced by Gonyaulax catenella in axenic culture. Biochemistry 5,1191 . Sort, H. and MBY~x, K. F. (1937) Paralytic shellfish poisoning. A.M.A . Arch . Path . 24, 560. Sonsngx, H., WHeooN, W. F., KOPOro, C. A. and Sro~t, R. (1937) Relation of paralytic shellfish poison to certain plankton organisms of the genus Gonyaulax. A.M.A . Arch . Path . 24, 537 . Wsn., C. S. (1952) Tables for convenient calculation of mediaa-effective dose (c.n,~ or sn~,) and instructions in their use. Biometrics S, 249.