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PubMed1982

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↗
PubMed1986

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1990

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↗
PubMed1991

Diarrhetic shellfish toxins: improvement of sample clean-up for HPLC determination.

Okadaic acid and dinophysistoxin-1, the principal toxic components in diarrhetic shellfish poisoning, may be detected by high-performance liquid chromatography and fluorometric measurement as 9-anthrylmethyl esters. However, "greasy" samples may occur and the fluorescent reagent 9-anthryldiazomethane may decompose during storage, resulting in impurities that may seriously interfere with quantitative determination. Ultrasonic treatment of the samples during derivatization with 9-anthryldiazomethane was found to improve reproducibility. This may result from increased access to reactive sites on toxins by 9-anthryldiazomethane due to disruption of micelles formed by toxins and other partly hydrophobic compounds. A procedure for cleaning the derivatized samples, using a 0.1 g silica cartridge column and different eluent compositions from that reported by LEE et al. (1987), was found to facilitate chromatogram interpretation. Deoxycholic acid, a commercial available bile acid, was found to be an acceptable internal standard. The 9-anthrylmethyl esters of okadaic acid, dinophysistoxin-1 and deoxycholic acid, were stable at 4 degrees C for at least seven days when stored dry or in methanol.

Animals↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1994

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↗
PubMed1992

An evaluation of the mouse bioassay applied to extracts of 'diarrhoetic' shellfish toxins.

The standard mouse bioassay, used to assess 'diarrhoetic shellfish poison' (DSP), is based on intraperitoneal administration of toxic mussel extracts, and monitoring of survival time within a 24-hr period. Toxic effects on mice were examined for extracts of mussel samples from two different regions of south Norway known to possess toxins of specific properties. Both samples revealed an exponential pattern in the dose-response relationship. Whereas the time lag from injection to death was linearly dependent on mouse weight, the effect of weight also increased with decreased sample toxicity. When tested with doses adjusted for weight, a marked individual variation was found within all size groups of mice. The results imply that, regarding prohibition limits for distribution and sale of mussels, a certain degree of variation with regard to time should be accepted in the testing of parallel samples. On the basis of the results, a revised method for the determination of toxicity by mouse bioassay is proposed for DSP testing. The method is based on administration to two mice of size-adjusted doses of extracts, followed by a 4-hr surveillance period and a 1-hr upper limit of acceptable time variation between parallel samples. The method shows advantages regarding savings of time and money, in precision in determination of toxicity level, as well as curtailed exposure to toxin and reduced suffering of laboratory animals.

Animals↗
PubMed1995

Induction of morphological transformation in BALB/3T3 mouse embryo cells by okadaic acid.

Okadaic acid is produced by several types of dinoflagellates (marine plankton) and has been implicated as a causative agent of diarrhoetic shellfish poisoning. Okadaic acid, a known tumour promoter in vivo, has been shown to promote morphological transformation of carcinogen-initiated BALB/3T3 cells. This study shows that okadaic acid is capable of inducing morphological transformation of BALB/3T3 cells in the absence of an initiator.

3T3 Cells↗
PubMed1994

Method for the identification of saxitoxin in rat urine.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.

Animals↗
PubMed2004

Morphological abnormalities and sensorimotor deficits in larval fish exposed to dissolved saxitoxin.

The dietary uptake of one suite of dinoflagellate-produced neurotoxins, that are commonly called paralytic shellfish poisoning (PSP) toxins, is known to cause acute fish kills. However, little is known about the effects of dissolved phase exposure and the potential sublethal effects of this route of exposure on early developmental stages of fish. Toxin exposure during early development is of particular concern because the embryos and larvae of some marine fish species may be unable to actively avoid the dissolved toxins that algal cells release into the water column during harmful algal blooms. Here we use the zebrafish (Danio rerio) as a model experimental system to explore the sublethal effects of a dissolved PSP toxin, saxitoxin (STX), on early development in fish, including sensorimotor function, morphology, and long-term growth and survival. Aqueous phase exposures of 229 +/- 7 microg STX eq. l(-1) caused reductions in sensorimotor function as early as 48 h postfertilization (hpf) and paralysis in all larvae by 4 days postfertilization (dpf). Rohon-Beard mechanosensory neurons appeared to be more sensitive to STX than dorsal root ganglion neurons at this dose. Additionally, exposure to 481 +/- 40 microg STX eq. l(-1) resulted in severe edema of the eye, pericardium, and yolk sac in all exposed larvae by 6 dpf. The onset of paralysis in STX-exposed larvae was stage-specific, with older larvae becoming paralyzed more quickly than younger larvae (5 h at 6 dpf as compared to 8 and 46 h for 4 and 2 dpf larvae, respectively). When transferred to clean water, many larvae recovered from the morphological and sensorimotor effects of STX. Thus, the sublethal effects of the toxin on larval morphology and behavior were reversible. However, zebrafish exposed to STX transiently during larval development (from 2 to 4 dpf) had significantly reduced growth and survival at 18 and 30 days of age. Collectively, these data show that (1) dissolved phase STX is bioavailable to fish embryos and larvae, (2) the toxin is a paralytic with potencies that are stage-specific for fish larvae, (3) the observed toxicological effects of STX exposure are reversible, and (4) a short-term toxin exposure can negatively impact the survival of fish several weeks later. Dissolved algal toxins may therefore have important sublethal effects on vulnerable species of fish.

Animals↗
PubMed2004

Evidence for differences in the metabolism of saxitoxin and C1+2 toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3.

The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1+2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 microg l(-1). Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 microg l(-1) CAM increased C1+2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1+2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1+2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3.

Agmatine↗
PubMed2005

Oligonucleotide quartz crystal microbalance sensor for the microalgae Alexandrium minutum (Dinophyceae).

We report the immobilization on a gold surface of a 20-base DNA probe labeled with disulfide group and on the selective hybridization with the complementary 20-base DNA strand. The oligonucleotide probe is the complementary strand of a partial sequence of the gene encoding for a large ribosomal RNA sub-unit which is a coding sequence of Alexandrium minutum DNA, a microalgae that produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts. The kinetics of DNA probe immobilization and hybridization were monitored in situ by using a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the setup is stable to within a few hertz, corresponding to the nanogram scale, for 3h and makes it possible to follow frequency change from immobilization of the probe to hybridization of the complementary DNA target. This setup constitutes a biosensor, which is sensitive and selective, and the hybridization ratio between hybridized complementary DNA and immobilized DNA probes is 47%.

Animals↗
PubMed2005

First report of aphantoxins in China--waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi.

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flos-aquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flos-aquae in China.

Animals↗
PubMed2006

Comparative gene expression of PSP-toxin producing and non-toxic Anabaena circinalis strains.

Blooms of the freshwater cyanobacterium Anabaena circinalis are recognised as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives, also known as paralytic shellfish poisoning (PSP) toxins. In this study the transcriptional profile of PSP toxin-producing and non-toxic strains of A. circinalis was investigated by means of a DNA microarray approach. Additionally, gene expression was studied after exposure of toxic A. circinalis cultures to lidocaine hydrochloride at 1 microM for 2 h. Under standard growth conditions, a limited number of putative toxic-strain distinctive DNA fragments, identified in previous studies, were preferentially expressed in toxic versus non-toxic strains. The same genes did not significantly change their expression after exposure to 1 microM lidocaine, conditions previously shown to induce STX production in the cyanobacterium Cylindrospermopsis raciborskii T3. Lidocaine supplementation, however, enhanced the transcription of genes involved in physiological adaptive responses and bloom formation in cyanobacteria, such as the gas vesicle structural protein A and phycocyanin. The heat shock protein HSP-70 and the chlorophyll-a binding protein isiA were significantly repressed by lidocaine exposure. Stress response proteins and genes implicated in secondary metabolism were repressed, including phosphopantetheinyl transferases. The BGGM1 DNA microarray, used in this study, was shown to be suitable for gene expression studies in cultured toxic cyanobacteria and allowed the analysis of gene transcripts associated with surface scum formation by toxic A. circinalis.

Anabaena↗
PubMed2006

The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal.

Animals↗