Toxleon, 1977, Vol. 13, pp" 4319. Peraemon Preis. Prlnbed In Greet Brtteln.
NEUROTOXINS FROM THREE SPECIES OF CALIFORNIA GOBY : CLEVELANDIA IOS, ACANTHOGOBIUS FLAVIMANUS AND GILLICHTHYS MIRABILIS
K. SHERYL ELAM, FhEDERICK A. ~THRMAN Hopkins Marine Station, Pacific Grove, California 93950, U.S.A . and the Department of Physiology, Stanford University, Stanford, California, U.S.A . and
YoxG H. KIM and HA1tRY S. MosHBx Department of Chemistry, Stanford University, Stanford, California, U.S.A . (Acceptedjar publkation 13 July 197 K. S. Et~1, F. A. Fvi~+ex, Y. H. K1us and H. S. MosxEts. Neurotoxins from three species of California goby : Clevelandla ios, Acanthogobius Jlavimonus and Glllichthys mlrabills. Textron 15, 45-49, 1977 .-Three species of fish, Clevelandla ios, Aconthogoblus flavtmanus, and Gilllchthys mlrabilis, belonging to the family Gobüdae were found to contain toxic substances that were lethal in mice and blocked the compound action potential of desheathed bullfrog axons. In G. »rirabills the toxin was found principally in the ovaries and liver. The toxin from the ovaries was purified and found to have a retention time by liquid chromatography similar to that of tetrodotoxin . From NMR spectroscopy theidentity with tetrodotoxin could not be excluded owing to the small size of the sample. However, newts (Taricha granulosa) that are extt+emely resistant to tetrodotoxin were killed by moderate doses of the toxin from Gilllchthys ovaries, indicating that it differs from tetrodotoxin. INTRODUCTION
and NOGUCHI (1971) were first to describe a tetrodotoxin-like substance in a non-tetraodontiform fish, the goby Gobies criniger. Tetrodotoxin is the potent neurotoxin originally known from pufferfishes of the order Tetraodontiformes and later found to be identical to tarichatoxin from newts of the genus Taricha (BUCHWALD et aL, 1964). It was later confirmed as the toxic compound in Gobies criniger (NOGUCHI and HasHnIO~ro, 1973). Tetrodotoxin has been identified in three species of Central and South American arrow poison frogs of the family Atelopidae, and a closely related new toxin (chiriquitoxin) was found in one of these (KIM et al., 1975). In addition, the neurotoxin from Octopus (Hapalochlaena) maculosus resembles tetrodotoxin in chemical and pharmacological properties (JARVIS et al., 1975). The discovery of tetrodotoxin and related substances in such diverse animal groups as these suggests that these compounds may be even more widespread than previously thought and may serve some physiological function other than, or in addition to, protection from predators. In order to determine whether tetrodotoxin and/or similar compounds might be found in species of Gobüdae other than Gobies criniger, three species were investigated in the work described here . The arrow goby Clevelandia ios Jordan and Gilbert, the Oriental goby Acanthogobius flavimartus Temminck and Schlegel and the longjaw mudsucker Gillichthys mirabilis Cooper were chosen for study because they were among the most common species of Gobüdae found in sloughs and shallow bays along the California coast. HASHIMOTO
45
46
K. 3. EL.AM, F. A. FUHRMAN, Y. H. KIM and H. S. MOSHER
We are not aware that any of these three species or others of the perhaps 500 species of Gobüdae except Gobius crnriger has been suspected of containing netuotoxins. MATERIALS AND METHODS
Collection ofspecimens Specimens of Clevelandta tos, a small species (overall length to 5 cm), were collected in July and August, 1974, at Elkhom Slough, Monterey County . Specimens of Acanthogobtus Jlavimanus (length to 23 cm) were
collected in November, 1974, at Berkeley Aquatic Park, a shallow basin with tidal flow from San Francisco Bay. Specimens of Gillichthys mirabilis (species length to 20 cm), used as bait by sports fishermen, were purchased in February, 1975, in San Diego County . In every case, specimens were brought to the laboratory and kept alive in circulating seawater tanks until use.
Extraction and purification procedures Batches of whole Clevelandia tos were used since this species was too small for separation of tissues to be feasible . Specimens of Acanthogobius Jfavimmws and Gt/lichthys mirabilis were separated by sex based upon internal examination . The gonads, liver and gallbladder were removed from each . The tissues of Acanthogobius fiavimartus were extracted in the following batches : female ovary (non-gravid), liver and gallbladder ;
remainder of female body tissues; male testes, liver and gallbladder ; remainder of male body tissues. The tissues of Gillichthys mirabilis were extracted in the following batches: female ovary (gravid) ; female liver and gallbladder ; remainder of female body tissues ; male liver and gallbladder ; remainder of male tissues. As the original purpose of the investigation was to ascertain whether or not these species contained tetrodotoxin or tetrodotoxin-1~7ce compounds, tho extraction and purification procedures used were those known to be effective for tetrodotoxin . Each batch of fresh tissue was homogenized in a Waring blender, brought to pH 3 with acetic acid and extracted twice at 1-Z°C for a total of 48 hr with methanol. After centrifugation, the methanol was removed from the supernatant on a flashevaporator at 40°C. The residue was dissolved in distilledwater and dialyzed through Spectrapor No. 2 membrane tubing, mol. wt cut-off: 12,000-14,000 (Spechvm Medical Industries, Inc.), at C>-10°C for 24 hr twice. The combined dialysates for each batch were lyophilized, and the dried residue was dissolved in distilled water. Extnicts from Clevelandta tos and Gillichthys mtrabilis were further purified by ion exchange chromatography according to procedures for tetrodotoxin from Goro et al. (196 and Nocucrtr and H~saro (1973) . The dissolved dialysate from each batch was placed on a 1 x 10 cm column of Amberlite IRC-50 (Mallincrodt Chemical Co .) converted to the NH,* form . The column was washed with distilled water and eluted with 1 M acetic acid. The first 50 ml acid fraction was lyophilized, and the dried product was dissolved in distilled water. Only eluates from tho extraction of Gilltchrhys mtrabilis ovaries were further purified . They were added to a column of Sephadex G-10 (Pharnracia Fine Chemicals, Inc.) with a bed volume of 2 ml and length of 20 cm in a small amount of pyridine--acetate buffer (pyridine-acetic acid-distilled water, 12 : 1 : S00) . The column was eluted with the pyridine-acetate buffer under forced air pressure, and the 0"~2 "S ml fraction of the elutant was lyophilized. The residue was dissolved in 1"S ~ acetic acid-methanol, l : 3, and filtered through Whatman No. 42 paper. Bioassay
Initially, mice were used for bioassay by i.p . injection of small volumes of tissue extracts at various stages of purification . One mouse unit (MLJ) is defined as the amount required to kill a 20 g mouse in 10 min after i.p. injection. Because a mouse assay requires a large amount of material, it was abandoned for routine use in favor of a nern assay using procedures modified from STxoxa et al. (1973). The products were diluted with Ringer's solution and assayed on bullfrog (Reno catesbtana) sciatic nerve preparations which were desheathed in their midsections. Each nerve was placed across silver electrodes in a moist chamber and stimulated with shocks from a Grass Model S-4B stimulator equipped with a photoelectric stimulus isolation unit. An oscilloscope (Tektronix 5103N) was used to monitor the height of the compound action potential picked up with a second pair of silver electrodes. A plastic cup was placed between the stimulating and recording electrodes and contained 0"8 ml Ringer's solution, including any test dose of either pure tetrodotoxin or purified goby extract, and the desheathed 14- to 16-mm midsection of the nern . A standard curve was derived using purified tetrodotoxin (Research Laboratories of Sankyo Co., Tokyo) . The equation obtained by linear regression analysis, x = (y + 2"41)/2 "55, where x is the logarithm of the dosage of tetrodotoxin in ng perml and y is the percent reduction in the height of the compound action potential expressed in probits, was used to calculate the amount of tetrodotoxin (or some other neurotoxic constituent in terms of equivalent tetrodotoxin activity) in an extract of unknown wnoentration from its effect on a nerve. Chemistry
Eluates of ovarian extracts of Gillichthys mirabilis from Sepbadex columns in pyridine acetate buffer were lyoph7ized three times under high vacuum . The residue was dissolved in D,O-CD,CO,D for NMR studies, which were inconclusive . The same sample was then chromatographed at 250 p .s.i . in a Bio-Gel P-2 column using pyridine acetate buffer at pH 6"2 as desarUed for tetrodotoxin (Knit et al., 197 . Eluant fractions were combined in groups and tested for toxicity on mice, and the NMR studies repeated .
Goby Neurotoxins
47
RESULTS
Bioassay Extracts of Clevelandaa tos partially purified by dialysis produced hindleg paralysis and convulsions-symptoms characteristic of tetrodotoxin-and death within 30 min in mice injected intraperitoneally with doses equivalent to 29 g of fish tissue . Extracts further purified by chromatography on IRG50 also produced the same symptoms and death in mice, and abolished the compound action potential of bullfrog sciatic nerves as well (Table 1). Nevertheless, the toxicity amounted to only about 1 MU per fish, and because of their small size (0~3 g per fish) this species was not studied further. TAHLE 1 . BIOASSAY OF EXTRACTS OP GORY TIS4LfPS BY BLOCKING ACTION POTENTIALS IN AULLFRO(3 SCIATIC NERVES
Species
Tissue
C.levelandla ios
Whole fish
Aconthogoblusflavimanus
Female liver and non-gravid ovaries
Gillichthys mlrabills
Gravid ovaries Female liver Remainder of female tissues Male liver Remainder of male tissues
Weight (g) 250'
Potency expressed as pg of tetrodotoxin per g tiss ue 0~4
S~S
6'1
172
10 200
6-6
2548 14-6
NEUROTOXINS FROM THREE SPECIES OF CALIFORNIA GOBY : CLEVELANDIA IOS, ACANTHOGOBIUS FLAVIMANUS AND GILLICHTHYS MIRABILIS
K. SHERYL ELAM, FhEDERICK A. ~THRMAN Hopkins Marine Station, Pacific Grove, California 93950, U.S.A . and the Department of Physiology, Stanford University, Stanford, California, U.S.A . and
YoxG H. KIM and HA1tRY S. MosHBx Department of Chemistry, Stanford University, Stanford, California, U.S.A . (Acceptedjar publkation 13 July 197 K. S. Et~1, F. A. Fvi~+ex, Y. H. K1us and H. S. MosxEts. Neurotoxins from three species of California goby : Clevelandla ios, Acanthogobius Jlavimonus and Glllichthys mlrabills. Textron 15, 45-49, 1977 .-Three species of fish, Clevelandla ios, Aconthogoblus flavtmanus, and Gilllchthys mlrabilis, belonging to the family Gobüdae were found to contain toxic substances that were lethal in mice and blocked the compound action potential of desheathed bullfrog axons. In G. »rirabills the toxin was found principally in the ovaries and liver. The toxin from the ovaries was purified and found to have a retention time by liquid chromatography similar to that of tetrodotoxin . From NMR spectroscopy theidentity with tetrodotoxin could not be excluded owing to the small size of the sample. However, newts (Taricha granulosa) that are extt+emely resistant to tetrodotoxin were killed by moderate doses of the toxin from Gilllchthys ovaries, indicating that it differs from tetrodotoxin. INTRODUCTION
and NOGUCHI (1971) were first to describe a tetrodotoxin-like substance in a non-tetraodontiform fish, the goby Gobies criniger. Tetrodotoxin is the potent neurotoxin originally known from pufferfishes of the order Tetraodontiformes and later found to be identical to tarichatoxin from newts of the genus Taricha (BUCHWALD et aL, 1964). It was later confirmed as the toxic compound in Gobies criniger (NOGUCHI and HasHnIO~ro, 1973). Tetrodotoxin has been identified in three species of Central and South American arrow poison frogs of the family Atelopidae, and a closely related new toxin (chiriquitoxin) was found in one of these (KIM et al., 1975). In addition, the neurotoxin from Octopus (Hapalochlaena) maculosus resembles tetrodotoxin in chemical and pharmacological properties (JARVIS et al., 1975). The discovery of tetrodotoxin and related substances in such diverse animal groups as these suggests that these compounds may be even more widespread than previously thought and may serve some physiological function other than, or in addition to, protection from predators. In order to determine whether tetrodotoxin and/or similar compounds might be found in species of Gobüdae other than Gobies criniger, three species were investigated in the work described here . The arrow goby Clevelandia ios Jordan and Gilbert, the Oriental goby Acanthogobius flavimartus Temminck and Schlegel and the longjaw mudsucker Gillichthys mirabilis Cooper were chosen for study because they were among the most common species of Gobüdae found in sloughs and shallow bays along the California coast. HASHIMOTO
45
46
K. 3. EL.AM, F. A. FUHRMAN, Y. H. KIM and H. S. MOSHER
We are not aware that any of these three species or others of the perhaps 500 species of Gobüdae except Gobius crnriger has been suspected of containing netuotoxins. MATERIALS AND METHODS
Collection ofspecimens Specimens of Clevelandta tos, a small species (overall length to 5 cm), were collected in July and August, 1974, at Elkhom Slough, Monterey County . Specimens of Acanthogobtus Jlavimanus (length to 23 cm) were
collected in November, 1974, at Berkeley Aquatic Park, a shallow basin with tidal flow from San Francisco Bay. Specimens of Gillichthys mirabilis (species length to 20 cm), used as bait by sports fishermen, were purchased in February, 1975, in San Diego County . In every case, specimens were brought to the laboratory and kept alive in circulating seawater tanks until use.
Extraction and purification procedures Batches of whole Clevelandia tos were used since this species was too small for separation of tissues to be feasible . Specimens of Acanthogobius Jfavimmws and Gt/lichthys mirabilis were separated by sex based upon internal examination . The gonads, liver and gallbladder were removed from each . The tissues of Acanthogobius fiavimartus were extracted in the following batches : female ovary (non-gravid), liver and gallbladder ;
remainder of female body tissues; male testes, liver and gallbladder ; remainder of male body tissues. The tissues of Gillichthys mirabilis were extracted in the following batches: female ovary (gravid) ; female liver and gallbladder ; remainder of female body tissues ; male liver and gallbladder ; remainder of male tissues. As the original purpose of the investigation was to ascertain whether or not these species contained tetrodotoxin or tetrodotoxin-1~7ce compounds, tho extraction and purification procedures used were those known to be effective for tetrodotoxin . Each batch of fresh tissue was homogenized in a Waring blender, brought to pH 3 with acetic acid and extracted twice at 1-Z°C for a total of 48 hr with methanol. After centrifugation, the methanol was removed from the supernatant on a flashevaporator at 40°C. The residue was dissolved in distilledwater and dialyzed through Spectrapor No. 2 membrane tubing, mol. wt cut-off: 12,000-14,000 (Spechvm Medical Industries, Inc.), at C>-10°C for 24 hr twice. The combined dialysates for each batch were lyophilized, and the dried residue was dissolved in distilled water. Extnicts from Clevelandta tos and Gillichthys mtrabilis were further purified by ion exchange chromatography according to procedures for tetrodotoxin from Goro et al. (196 and Nocucrtr and H~saro (1973) . The dissolved dialysate from each batch was placed on a 1 x 10 cm column of Amberlite IRC-50 (Mallincrodt Chemical Co .) converted to the NH,* form . The column was washed with distilled water and eluted with 1 M acetic acid. The first 50 ml acid fraction was lyophilized, and the dried product was dissolved in distilled water. Only eluates from tho extraction of Gilltchrhys mtrabilis ovaries were further purified . They were added to a column of Sephadex G-10 (Pharnracia Fine Chemicals, Inc.) with a bed volume of 2 ml and length of 20 cm in a small amount of pyridine--acetate buffer (pyridine-acetic acid-distilled water, 12 : 1 : S00) . The column was eluted with the pyridine-acetate buffer under forced air pressure, and the 0"~2 "S ml fraction of the elutant was lyophilized. The residue was dissolved in 1"S ~ acetic acid-methanol, l : 3, and filtered through Whatman No. 42 paper. Bioassay
Initially, mice were used for bioassay by i.p . injection of small volumes of tissue extracts at various stages of purification . One mouse unit (MLJ) is defined as the amount required to kill a 20 g mouse in 10 min after i.p. injection. Because a mouse assay requires a large amount of material, it was abandoned for routine use in favor of a nern assay using procedures modified from STxoxa et al. (1973). The products were diluted with Ringer's solution and assayed on bullfrog (Reno catesbtana) sciatic nerve preparations which were desheathed in their midsections. Each nerve was placed across silver electrodes in a moist chamber and stimulated with shocks from a Grass Model S-4B stimulator equipped with a photoelectric stimulus isolation unit. An oscilloscope (Tektronix 5103N) was used to monitor the height of the compound action potential picked up with a second pair of silver electrodes. A plastic cup was placed between the stimulating and recording electrodes and contained 0"8 ml Ringer's solution, including any test dose of either pure tetrodotoxin or purified goby extract, and the desheathed 14- to 16-mm midsection of the nern . A standard curve was derived using purified tetrodotoxin (Research Laboratories of Sankyo Co., Tokyo) . The equation obtained by linear regression analysis, x = (y + 2"41)/2 "55, where x is the logarithm of the dosage of tetrodotoxin in ng perml and y is the percent reduction in the height of the compound action potential expressed in probits, was used to calculate the amount of tetrodotoxin (or some other neurotoxic constituent in terms of equivalent tetrodotoxin activity) in an extract of unknown wnoentration from its effect on a nerve. Chemistry
Eluates of ovarian extracts of Gillichthys mirabilis from Sepbadex columns in pyridine acetate buffer were lyoph7ized three times under high vacuum . The residue was dissolved in D,O-CD,CO,D for NMR studies, which were inconclusive . The same sample was then chromatographed at 250 p .s.i . in a Bio-Gel P-2 column using pyridine acetate buffer at pH 6"2 as desarUed for tetrodotoxin (Knit et al., 197 . Eluant fractions were combined in groups and tested for toxicity on mice, and the NMR studies repeated .
Goby Neurotoxins
47
RESULTS
Bioassay Extracts of Clevelandaa tos partially purified by dialysis produced hindleg paralysis and convulsions-symptoms characteristic of tetrodotoxin-and death within 30 min in mice injected intraperitoneally with doses equivalent to 29 g of fish tissue . Extracts further purified by chromatography on IRG50 also produced the same symptoms and death in mice, and abolished the compound action potential of bullfrog sciatic nerves as well (Table 1). Nevertheless, the toxicity amounted to only about 1 MU per fish, and because of their small size (0~3 g per fish) this species was not studied further. TAHLE 1 . BIOASSAY OF EXTRACTS OP GORY TIS4LfPS BY BLOCKING ACTION POTENTIALS IN AULLFRO(3 SCIATIC NERVES
Species
Tissue
C.levelandla ios
Whole fish
Aconthogoblusflavimanus
Female liver and non-gravid ovaries
Gillichthys mlrabills
Gravid ovaries Female liver Remainder of female tissues Male liver Remainder of male tissues
Weight (g) 250'
Potency expressed as pg of tetrodotoxin per g tiss ue 0~4
S~S
6'1
172
10 200
6-6
2548 14-6
