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Branched long-chain bases from the bivalve Corbicula sandai.

Long-chain bases were liberated from a crude mixture of sphingolipids from whole tissue of the fresh-water bivalve C. sandai, and conversion of the bases into N-acetyl-0-trimethylsily derivatives was accomplished. The derivatized bases were analyzed by combined gas-liquid chromatography and mass spectrometry. A portion of the sphingolipids was subjected to catalytic hydrogenation from whch saturated long-chain bases (sphinganines) were obtained. The saturated bases were oxidized with lead tetra-acetate and the aldehydes produced were analyzed by gas-liquid chromatography. The aldehydes were further oxidized to acids with silver oxide, the resulting fatty acids methylated and also analyzed by gas-liquid chromatography. By these analyses, altogether five long-chain bases were identified, consisting of hexadeca-4-sphingenine (15%), heptadeca-4-sphingenine (2%), iso-octadeca-4-sphingenine (13%), octadeca-4-sphingenine (39%) and anteiso-noadeca-4-sphingenine (31%). So far no branches have been found in shellfish spingolipid long-chain bases.

Animals↗

Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis.

The purification and crystallization of T17, a toxin from Ptychodiscus brevis, is reported. The toxicity of this compound and a second toxin known as T34 are compared by i.v., i.p. and oral administration in mice. Both toxins produce symptoms characteristic of muscarnic stimulants; hypersalivation, rhinorrhea and excessive urination and defecation being the most commonly observed. T17, which is orally toxic, is believed to be the agent responsible for Neurotoxic Shellfish Poisoning.

Administration, Oral↗

Comparison of the toxins of the blue-green alga Aphanizomenon flos-aquae with the Gonyaulax toxins.

A toxic strain of Aphanizomenon flos-aquae (NH-1), isolated from a toxic bloom in a pond in Durham, New Hampshire, has been mass cultured in the laboratory. The toxin was extracted by ultrasonic disruption of the cells and purified by; (a) filtration through a 10 kilodalton filter, and (b) chromatography on a strong cation exchange resin column using 0.01 M, then 0.1 M, pH 5, sodium acetate buffer followed by 0.75 M hydrochloric acid. Mouse assays and fluorescence generated by hydrogen peroxide oxidation were used to monitor the fractions. Only a nonfluorescent toxic peak followed immediately by a fluorescent less-toxic peak were detected, both eluting with the hydrochloric acid fractions. The toxins were identical in behavior to neosaxitoxin and saxitoxin, respectively, when compared with elution profiles of the paralytic shellfish poisons from Gonyaulax tamarensis var. excavata and by paper electrophoretic and thin-layer chromatographic comparisons. The toxin profile appears to be different from that of a previously isolated strain of A. flos-aquae from Kezar Lake.

Animals↗

Occurrence of saxitoxin in puffer fish.

Three species of puffer fish, Takifugu poecilonotus, T. vermicularis and T. radiatus, were examined for the presence of toxic components other than tetrodotoxin. Saxitoxin, a paralytic shellfish toxin, was found in the livers, ovaries and digestive tracts of the first two species but not in the last species. The puffers are assumed to accumulate saxitoxin by feeding on bivalves that have ingested a toxic dinoflagellate Protogonyaulax tamarensis. Another toxic component is present in the liver of T. poecilonotus, but its structural relationship to tetrodotoxin or saxitoxin is questionable.

Animals↗

Assessment of potential activation of gonyautoxin V in the stomach of mice and rats.

Transformation of gonyautoxin V, a paralytic shellfish toxin of low lethality, to highly potent saxitoxin in the stomach was assessed by incubation in gastric juices. Only 8-15% of the toxin was converted to saxitoxin under drastic conditions. Comparison of oral and i.p. LD50 values of both toxins indicated that little conversion took place in the stomach of mice.

Animals↗

Occurrence of a water soluble toxin in a parrotfish (Ypsiscarus ovifrons) which is probably responsible for parrotfish liver poisoning.

The liver of the parrotfish Ypsiscarus ovifrons sometimes causes severe muscle pain, paralysis and dyspnea when ingested by humans. Individual livers, ovaries and digestive tracts and their contents were examined for lethal potency in mice. They were all toxic, except for livers obtained from April to June. Lethal potency ranged from 0.25 to 5.0 MU/g tissue. Livers were extracted with acidic aqueous ethanol and the extracts purified by charcoal treatment, gel filtration and partition and reversed phase column chromatography. Both the crude and partially purified toxin showed chemical and/or pharmacological properties different from those of tetrodotoxin or paralytic shellfish poisons.

Animals↗

Local differences in toxin composition of a xanthid crab Atergatis floridus inhabiting Ishigaki Island, Okinawa.

Specimens of a xanthid crab Atergatis floridus were collected from two adjacent areas in Kabira Bay, Ishigaki Island, Okinawa and compared with respect to toxicity and toxin composition. 'Reef specimens', which were collected from the reefs of Kabira Bay, showed an average toxicity score of 380 MU/g as paralytic shellfish poison, and a toxin composition consisting mainly of saxitoxin and a neosaxitoxin-related substance. On the other hand, 'Kojima specimens', which were collected from a small island in the bay, showed an average toxicity of 38 MU/g as tetrodotoxin, and a toxin composition consisting mainly of tetrodotoxin and related substance(s).

Animals↗

A tissue culture assay for tetrodotoxin, saxitoxin and related toxins.

In the presence of ouabain, veratridine enhances sodium influx in the mouse neuroblastoma cell line Neuro-2A (ATCC, CCL131), causing cellular swelling and subsequent death. Tetrodotoxin (puffer fish toxin) or saxitoxin (paralytic shellfish poison), both of which block the sodium channel of excitable membranes, antagonize this effect, enabling cell growth to continue. This phenomenon was used as the basis of a new assay for these toxins. It is also possible to estimate the quantity of TTX from the relationship between TTX concentration and percentage of living cells. This new method is simple, inexpensive, and sensitive, and may replace the conventional mouse bioassay.

Animals↗

Appearance and partial purification of a high molecular weight protein in crabs exposed to saxitoxin.

This paper provides evidence for a protein component which appears to be involved in the seasonal resistance of small shore crabs, Hemigrapsus oregonesis and Hemigrapsus nudus to saxitoxin, a principle neurotoxin involved in paralytic shellfish poisoning (PSP). This unique protein complex was isolated and partially purified by ion exchange chromatography using DEAE-cellulose from visceral tissue extracts of resistant crabs. The complex was absent in control crabs that were sensitive to saxitoxin. In addition, the protein complex was induced in the crab after acute administration of low doses of saxitoxin. Results indicate that the protein complex is acidic in nature and has an apparent mol. wt of 145,000.

Animals↗

Toxins extracted from Australian specimens of the crab, Eriphia sebana (Xanthidae).

Aqueous extracts of 75 specimens of the xanthid crab, Eriphia sebana, collected from coral reefs in the Capricorn Group, Great Barrier Reef, Australia, were assayed for toxicity to mice. Thirty-five extracts contained detectable toxicity. Toxicity was quantified by i.p. injection of mice where 1 mouse unit (MU) was defined as the amount of toxin required to kill a 20 g mouse in 15 min. Twenty-four of the extracts induced signs in mice similar to signs expected if paralytic shellfish toxins (PSTs) or tetrodotoxin were the predominant toxins. The highest toxicity found was 887.4 MU from a 98.6 g crab (= 9.0 MU/g). Purification of five combined groups of extracts from crabs collected at the same time and area revealed for the first time the presence in E. sebana of toxins similar to PSTs. TLC and electrophoretic studies showed that these toxins behaved like saxitoxin, neosaxitoxin, gonyautoxin-1 and gonyautoxin-2. Two of these groups of extracts contained only one of these PST-like toxins and the remainder contained two. Eleven of the extracts caused signs in injected mice dissimilar to signs induced by PSTs or tetrodotoxin. Foregut contents of 33 crabs contained fish, crustacean and algal remains. Crustacean remains in one gut content sample included carapace fragments bearing distinctive surface features found on the smaller toxic xanthids, Actaeodes tomentosus and Pilodius areolatus. The combined gut contents of these crabs weighed 10.4 g and the aqueous extract of which contained 4.4 MU/g of toxin, supporting the suggestion of a probable dietary origin of the toxins present in the crabs.

Animals↗

Relationship between domoic acid levels in the blue mussel (Mytilus edulis) and toxicity in mice.

Monitoring of eastern blue mussels (Mytilus edulis), contaminated with domoic acid, involved mouse bioassays and quantitative analysis using HPLC. Mice undergo a typical scratching syndrome at sublethal as well as lethal doses of domoic acid. The onset of scratching behaviour and time of death in mice were inversely related to the dosage of domoic acid. An LD50 (i.p.) of 3.6 mg domoic acid/kg mouse was calculated. Toxic mussels held in tanks and flushed with uncontaminated sea water showed a decline in domoic acid concentration in mussel tissue with time. In addition, domoic acid concentrations in mussels from two infected rivers declined to negligible levels in 40-50 days under normal environmental conditions. The bulk of domoic acid and toxicity was located in the hepatopancreas which also contained large amounts of chlorophyll-A, an algae biomass indicator, relative to control mussels. These results support the conclusion that domoic acid was the primary causative factor in the shellfish poisonings from Prince Edward Island mussels in late 1987.

Animals↗

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.

Animals↗

Toxin composition of resting cysts of Alexandrium tamarense (Dinophyceae).

Paralytic shellfish toxin composition in the resting cysts of the dinoflagellate Alexandrium tamarense was investigated by means of high performance liquid chromatography. A comparison was made between cysts collected from ship ballast tank sediments, natural population of motile vegetative cells collected from the area where ballast water was taken, as well as cultured vegetative cells established from the cysts and the natural plankton bloom. Total toxin concentration of the cysts (595 fmole/cell) was six-fold higher than that of the natural population of vegetative cells. They contained the same ten toxic components but in different relative abundances. The higher proportion of 11-alpha-hydroxysulfate epimers in the cysts suggests that the biosynthesis of toxins is halted at an early stage in cyst formation.

Animals↗

Short-time cytotoxicity of mussel extracts: a new bioassay for okadaic acid detection.

Okadaic acid (OA), the main toxin responsible for diarrhoeic shellfish poisoning (DSP) has high cytotoxicity for KB cell cultures (apparent after 3 hr of contact), facilitating rapid detection in contaminated mussels. We developed a method to determine the minimal active concentration (MAC) based on direct microscopic study of toxin-induced changes in cell morphology. A high correlation was found between the MAC of tested extracts and corresponding OA concentrations in mussel hepatopancreas as measured by high performance liquid chromatography. This technique is rapid and reproducible and does not require the use of living animals.

Animals↗

Actions of decarbamoyloxysaxitoxin and decarbamoylneosaxitoxin on the frog skeletal muscle fiber.

Two new analogues of the decarbamoyl series of paralytic shellfish toxins have been isolated through improved HPLC methods. In decarbamoyloxysaxitoxin (doSTX), the -OH function at C-13 of decarbamoylsaxitoxin (dcSTX) is changed to -CH3. In decarbamoylneosaxitoxin (dcneoSTX), the carbomyl side-chain of neosaxitoxin (neoSTX) has been removed. The new analogues were assayed on voltage-clamped frog skeletal muscle fiber for their potency in reducing the sodium current. Compared with neoSTX, the relative potencies of dcneoSTX are: 0.003 (at pH 6.50), 0.004 (pH 7.25), and 0.005 (pH 8.25). The influence of pH on the potency is the same in neoSTX and dcneoSTX. The fractional loss of potency caused by decarbamoylation is much greater in neoSTX than in STX, possibly because of an intramolecular interaction between the N-1 -OH in neoSTX and the -OH on C-13. Compared with STX, the ED50 for reducing INa by doSTX is 618 nM, making its relative potency 0.008 that of STX. Energetically, the decreased potency can be accounted for by the loss of two hydrogen bonds, one at the C-13 -OH of dcSTX, and the other at the amino group in the carbamoyl function of STX. These two groups resemble the C-6 and C-11 -OHs in tetrodotoxin, and probably bind to the same site-points. Thus, the near-identical actions of STX and TTX can be attributed to the common sharing of one ion-pair site and four hydrogen-bonding sites. If glutamate 387 of rat brain sodium channel II were the anionic site which ion-pairs with the 7, 8, 9 guanidinium of STX, then the carbonyl oxygen of asparagin 388 is the hydrogen-acceptor for the C-12 gem-diols.

Animals↗

Comparison of paralytic toxins in aquaculture of purple clam in Taiwan.

Food poisoning incidents due to ingesting the cultured purple clam Soletellina diphos occurred in western Taiwan in February 1991. Clam specimens, sediment and the dinoflagellate Alexandrium tamarensis were collected and assayed for lethality as paralytic shellfish poison (PSP). The lethality of purple clam, wet sediment and phytoplankton was 1700 mouse units per specimen (MU/specimen), 0.05 MU/g and 3.6 x 10(-5) MU/cell, respectively. The toxins obtained from clam, sediment and phytoplankton consisted of gonyautoxins 1-4, along with trace amounts of neosaxitoxin.

Animals↗

Effect of lyophilization on the stability of gonyautoxins obtained from contaminated mussels.

This study describes the stability of gonyautoxins (GTX) and C toxins obtained from contaminated mussels and stored at different temperatures in lyophilized samples. Analyses of extracts from mussels contaminated with paralytic shellfish poison (PSP) indicated the presence of gonyautoxins as the major component in red tides of the North-West coast of Spain. These GTX and C toxins were extracted from contaminated mussels (Mytilus galloprovincialis Lmk) and partially purified by chromatography on Bio-Gel P-2 and Bio-Rex 70. The stability of these toxins was analysed by high performance liquid chromatography. GTX 4 and GTX 6 are the most stable toxins among GTX. We conclude that the lyophilization procedure is not the safest way to process most of the gonyautoxins. However, the lyophilization procedure made the C toxins unstable, so clearly this procedure must be rejected.

Animals↗

Determination of okadaic acid content of dinoflagellate cells: a comparison of the HPLC-fluorescent method and two monoclonal antibody ELISA test kits.

Total okadaic acid (okadaic acid plus methylokadaic acid) in acclimated clones of the dinoflagellates Prorocentrum hoffmannianum and P. lima was determined using the HPLC-fluorescent method, UBE ELISA test kit, and Rougier ELISA test kit. The nonokadaic acid-producing species. Amphidinium klebsii, Prorocentrum mexicanum, P. micans, P. cassubicum, and Gambierdiscus toxicus were examined using the same methods of analysis. All three methods yielded consistent results for P. hoffmannianum which produces only okadaic acid. However, results of the three methods were not consistent for P. lima which produces both okadaic acid and methylokadaic acid. The UBE ELISA demonstrated little or no cross-reactivity with methylokadaic acid; whereas, the Rougier ELISA demonstrated varying degrees of cross-reactivity with that analog. Analyses of nonokadaic acid producing-species yielded negative results, with one exception. The Rougier ELISA demonstrated reactivity with extracts of G. toxicus. Since outbreaks of DSP may be caused by okadaic acid, methylokadaic acid, or a combination of these toxins, both ELISA kits may underestimate total toxin present in toxic shellfish.

Animals↗