ABSTRACT
Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.
Subject(s)
Calcium/metabolism , Ciguatoxins/toxicity , Cytosol/drug effects , Dinoflagellida/chemistry , Marine Toxins/toxicity , Actins/metabolism , Actins/ultrastructure , Cell Line, Tumor , Cytosol/metabolism , Cytosol/ultrastructure , HumansABSTRACT
Poisoning after eating puffer fish containing highly lethal tetrodotoxin (TTX) is widespread in Asia. In 2008, naïve inland populations in Bangladesh were exposed to cheap puffer fish sold on markets. In three outbreaks, 141 patients with history of puffer fish consumption were hospitalized. Symptoms of poisoning included perioral paraesthesia, tingling over the entire body, nausea and vomiting, dizziness, headache, abdominal pain and muscular paralysis of the limbs. Seventeen patients (12%) died from rapidly developing respiratory arrest. Blood and urine samples from 38 patients were analyzed using a TTX-specific enzyme-linked immunoassay (ELISA). Medium to high TTX levels were detected (1.7-13.7 ng/ml) in the blood of 27 patients. TTX was below detection level (< 1.6 ng/ml) in 11 blood samples but the toxin was detected in urine. Ten patients had blood levels above 9 ng/ml and developed paralysis; seven of these died. The remaining patients recovered with supportive treatment. High concentrations of TTX and its analogues 4-epiTTX and 4,9-anhydroTTX were also found in cooked puffer fish by post-column liquid chromatography-fluorescence detection. To prevent future instances of puffer fish poisoning of this magnitude, measures should be implemented to increase awareness, to control markets and to establish toxicological testing. To improve the management of this and other poisoning in Bangladesh, facilities for life-saving assisted ventilation and related training of healthcare personnel are urgently needed at all levels of the health system.
Subject(s)
Disease Outbreaks , Fishes, Poisonous , Paralysis/chemically induced , Respiratory Insufficiency/chemically induced , Tetraodontiformes , Tetrodotoxin/poisoning , Adolescent , Adult , Animals , Bangladesh/epidemiology , Child , Child, Preschool , Chromatography, Liquid , Enzyme-Linked Immunosorbent Assay , Female , Foodborne Diseases/mortality , Humans , Male , Middle Aged , Paralysis/mortality , Paralysis/physiopathology , Poverty Areas , Respiratory Insufficiency/mortality , Respiratory Insufficiency/physiopathology , Tetrodotoxin/analysis , Young AdultABSTRACT
Most common phycotoxin poisoning syndromes have important neurological symptoms. However, little is known of the cellular and molecular targets of many of the phycotoxins that produce those human intoxications. We explore the effect of representative toxins on the membrane potential in human neuroblastoma cells by using a fluorimetric assay. Results presented in this study demonstrate that maitotoxin, palytoxins, brevetoxins, and ciguatoxins triggered a dose-dependent membrane depolarization. Mechanisms responsible for the toxins-induced changes in membrane potential are always related to a direct action of the compounds on membrane ion fluxes. This initial screening of the phycotoxins effect is the starting point to lately develop functional methods of detection.
Subject(s)
Marine Toxins/pharmacology , Neurons/drug effects , Cell Line, Tumor , Humans , Membrane Potentials/drug effects , Neurons/pathology , Time FactorsABSTRACT
Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.
Subject(s)
Carrier Proteins/isolation & purification , Sodium Channels/isolation & purification , Amino Acid Sequence , Amphibian Proteins , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cloning, Molecular , DNA Primers , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Glycosylation , Molecular Sequence Data , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Saxitoxin/metabolism , Sequence Homology, Amino Acid , Sodium Channels/chemistry , Sodium Channels/genetics , Sodium Channels/metabolism , Takifugu , Tetrodotoxin/metabolismABSTRACT
Tetrodotoxin (TTX) and its analogue 6-epiTTX were detected in 11-12 specimens of the red-spotted newt, Notophthalmus viridescens, by a post-column fluorescent-HPLC system and by LC/MS in selected ion monitoring mode. TTX levels varied considerably among individuals from low (less than 0.15 microg TTX/g newt) to high concentrations (23.5 microg TTX/g newt), while 6-epiTTX was found to be a minor constituent in all specimens.
Subject(s)
Notophthalmus viridescens/metabolism , Tetrodotoxin/analysis , Animals , Chromatography, High Pressure LiquidABSTRACT
Tetrodotoxin (TTX), a powerful sodium channel blocker, usually exists as a mixture of its analogs (TTXs) in natural sources. Due to the structural variation, some analogs are difficult to detect using the postcolumn liquid chromatography-fluorescent detection (LC-FLD) system. Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) analysis of TTXs can be achieved by a combination of chromatography on a reversed-phase column with long carbon chains (C30) and the mobile phase containing an ion pair reagent (ammonium heptafluorobutyrate). The relationship between the amount of applied standard TTX and its peak area on the mass chromatogram (m/z 320) showed good linearity over a range of 50-1000 pmol. The detection limit for TTX in the selected ion monitoring (SIM) mode was estimated to be 0.7 pmol (signal to noise ratio: 2). The tandem mass spectrometry (MS/MS) scan for the fragment ions of eight TTXs arising from the molecular ions provided characteristic spectra, and the structures of the origins of the prominent fragment ions were proposed. The intense fragment ions of TTX and 11-deoxyTTX were applicable to LC/MS/MS operated in the selected reaction monitoring mode. This method might be useful for further identification of TTXs.
Subject(s)
Chromatography, Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Tetrodotoxin/chemistry , Animals , Female , Fishes/metabolism , Fluorescence , Models, Chemical , Molecular Structure , Ovary/metabolism , Sensitivity and Specificity , Spectrophotometry, UltravioletABSTRACT
Toxicities and tetrodotoxin distribution in tissues of five puffer fish species commonly found in the littoral of Baja California Peninsula, Mexico (Sphoeroides annulatus, S. lobatus, S. lispus, Arothron meleagris and Canthigaster punctatissima) were evaluated by bioassay and HPLC. The toxicities estimated as tetrodotoxin-equivalents of all species were more than 0.42 microg/g in at least one of the tissues tested, and the highest was found in S. lispus liver (130 microg/g).
Subject(s)
Fishes, Poisonous/metabolism , Tetrodotoxin/metabolism , Tetrodotoxin/toxicity , Animals , Biological Assay , Chromatography, High Pressure Liquid , Male , Mexico , Mice , Tissue Distribution , Toxicity TestsABSTRACT
The dissociation constants for (3)H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis have been estimated to be 190- and 460-fold, respectively, larger than those to corresponding membranes of rat, by a rapid filtration assay, while these values for (3)H-PbTx-3 have been estimated to be one-third and one-half of those to rat, respectively. We have obtained a cDNA, encoding an entire voltage-gated Na(+) channel alpha-subunit (fMNa1, 1880 residues) from skeletal muscle of F. pardalis by composition of the fragments obtained from cDNA library and RT-PCR products. In fMNa1 protein, the residues for ion-selective filter and voltage sensor and the charged residues in SS2 regions of domains I-IV were conserved, but the aromatic amino acid (Phe/Tyr), commonly located in the SS2 region of domain I of tetrodotoxin-sensitive Na(+) channels, was replaced by Asn. With this particular criterion, we propose that the fMNa1 protein is a tetrodotoxin-resistant Na(+) channel.
Subject(s)
Brain/metabolism , Marine Toxins/pharmacokinetics , Muscle, Skeletal/metabolism , Neuromuscular Blocking Agents/pharmacokinetics , Oxocins , Saxitoxin/pharmacokinetics , Sodium Channels/chemistry , Sodium Channels/metabolism , Synaptic Membranes/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Membrane/metabolism , Evolution, Molecular , Fishes , Humans , Kinetics , Molecular Sequence Data , Phylogeny , Radioligand Assay , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Sodium Channels/genetics , TritiumABSTRACT
Second generation analogs of polycavemoside A (2) possessing a side chain at C-15 different from that of the natural toxin have been synthesized. The in vivo toxicities of these new compounds (expressed as the minimal lethal dose) have been evaluated in mice (ip) and compared to 2, its aglycone (8), and polycavemoside B (9). The bioactivity profile of enynene 5 is particularly notable.
Subject(s)
Disaccharides/chemical synthesis , Disaccharides/toxicity , Macrolides/chemical synthesis , Macrolides/toxicity , Marine Toxins/chemical synthesis , Marine Toxins/toxicity , Animals , Male , Mice , Mice, Inbred Strains , Structure-Activity Relationship , Toxicity TestsABSTRACT
A novel 10,7-lactone type of tetrodotoxin analog, 5-deoxytetrodotoxin, was isolated from the puffer fish, Fugu poecilonotus, and its structure was assigned by spectroscopic methods.
Subject(s)
Tetrodotoxin/chemistry , Tetrodotoxin/isolation & purification , Animals , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Fishes , Magnetic Resonance Spectroscopy , Molecular Structure , Spectrometry, FluorescenceABSTRACT
The widely used sodium channel blocker tetrodotoxin (TTX) is a compound that has six hydroxyl residues at the C-4, C-6, C-8, C-9, C-10, and C-11 positions in addition to a guanidinium group, which is positively charged in biological pH range. Thirteen analogs of this toxin with structural modifications involving one or more of these hydroxyls were examined on their affinity to a rat brain membrane preparation, which is known to contain sodium channels abundantly. The equilibrium dissociation constants associated with the binding of TTX and its analogs to the sodium channels were estimated, from their ability to inhibit the binding of [3H]saxitoxin, as follows (in nM): TTX, 1.8; chiriquitoxin, 1.0; 11-oxoTTX, 1.5; 11-norTTX-6,6-diol, 1.6; 11-norTTX-6(S)-ol, 23; 11-norTTX-6(R)-ol, 31; 11-deoxyTTX, 37; 6-epiTTX, 39; 4-epiTTX, 68; 4,9-anhydroTTX, 180; TTX-8-O-hemisuccinate, >380; TTX-11-carboxylic acid, >2300; tetrodonic acid, >3600; 5,6,11-trideoxyTTX, >5000. The reduction of the affinity observed with the analogs involving reduction or translocation of the hydroxyls at C-6 and C-11 is indicative of the contribution of these residues to the binding to sodium channels as hydrogen bond donors. The especially large value of the dissociation constant for TTX-11-carboxylic acid is consistent with the idea that the C-11-hydroxyl forms a hydrogen bond with a carboxylic acid residue of the channel protein. The markedly low affinity of TTX-8-O-hemisuccinate may possibly be ascribable to intramolecular salt-bridge formation, which neutralizes the positive charge of the guanidinium group.
Subject(s)
Brain/metabolism , Synaptic Membranes/metabolism , Tetrodotoxin/analogs & derivatives , Tetrodotoxin/pharmacology , Animals , Binding, Competitive , Hydroxylation , Kinetics , Models, Molecular , Molecular Conformation , Molecular Structure , Rats , Saxitoxin/pharmacokinetics , Structure-Activity Relationship , Synapses/drug effects , Synapses/metabolism , Synaptic Membranes/drug effects , Tetrodotoxin/chemical synthesis , Tetrodotoxin/pharmacokineticsABSTRACT
Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na+ channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na+ channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na(+)-channel ligands.
Subject(s)
Muscle, Skeletal/metabolism , Sodium Channels/metabolism , Tetrodotoxin/analogs & derivatives , Tetrodotoxin/metabolism , Animals , Binding, Competitive , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Dose-Response Relationship, Drug , Electrophorus , Electrophysiology , Isotope Labeling , Magnetic Resonance Spectroscopy , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Oxidation-Reduction , Patch-Clamp Techniques , Protons , Rana temporaria , Sodium Channels/drug effects , Spectrometry, Mass, Fast Atom Bombardment , Tetrodotoxin/analysis , Tetrodotoxin/chemistry , Tetrodotoxin/toxicity , TritiumABSTRACT
In alcoholic extracts from museum samples of the toads Atelopus subornatus and A. peruensis tetrodotoxin and its analogues, 4-epitetrodotoxin and 4,9-anhydrotetrodotoxin were detected by fluorometric HPLC analysis, extending the list of amphibians containing this toxin. However, the toxin was not detected in samples of Melanophryniscus stelzneri, Dendrophryniscus minutus and Oreophrynella sp.
Subject(s)
Bufonidae , Tetrodotoxin/analysis , Animals , Chromatography, High Pressure Liquid , Skin/chemistryABSTRACT
The water-soluble toxin present in skin of Colostethus inguinalis (Dendrobatidae) was identified as tetrodotoxin by fluorometric HPLC analysis. The amount of tetrodotoxin per frog skin was estimated by HPLC, mouse toxicity, and inhibition of [3H]saxitoxin binding to brain membranes as 0.1 to 1.2 micrograms. Small amounts of anhydrotetrodotoxin and 4-epietrodotoxin also were present. Tetrodotoxin-like activity was not detected by inhibition of [3H]saxitoxin binding in other species of Colostethus nor in other dendrobatids (Aromobates, Dendrobates, Phyllobates). Tetrodotoxin-like activity was present in extracts of skin of five species of Atelopus (Bufonidae). HPLC analysis identified tetrodotoxin as the major toxic component in Atelopus spumarius and A. varius, as a minor component in A. spurrelli, and as a trace component in A. ignescens and A. zeteki. The major tetrodotoxin-like compounds in the last three species were not identified. Tetrodotoxin-like activity was not detected by inhibition of [3H]saxitoxin binding in skin extracts from three other genera of bufonids.
Subject(s)
Amphibian Venoms/toxicity , Anura , Bufonidae , Skin/chemistry , Tetrodotoxin/toxicity , Amphibian Venoms/analysis , Amphibian Venoms/isolation & purification , Animals , Chromatography, High Pressure Liquid , Drug Interactions , Guinea Pigs , Injections, Intraperitoneal , Male , Mice , Saxitoxin/metabolism , Skin/metabolism , Species Specificity , Tetrodotoxin/analysis , Tetrodotoxin/isolation & purificationABSTRACT
Tetrodotoxin and its analogues, 4-epitetrodotoxin and 4,9-anhydrotetrodotoxin, were detected in the toad Atelopus oxyrhynchus by HPLC analysis. The toxin and its analogues were still present in a specimen which lived 3.5 years in captivity.