The Cardiovascular and Neurotoxic Effects of the Venoms of Six Bony and Cartilaginous Fish Species.

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10-100 µg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 µg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

Relationship between food and venom production in the estuarine stonefish Synanceia horrida.

BACKGROUND: The potential costs of venom production may be significant to many marine venomous taxa. In general, the parameters that influence the rate of venom production are poorly understood, but seem to be related to feeding frequency. METHODS: This study examines the effects of starvation on venom profile and venom yield on the estuarine stonefish (Synanceia horrida). In total, the venom of eight stonefishes was tested under two feeding regimes. Over a four week period, one of the two groups underwent an episode of suspended feeding, while the other was fed on a daily basis. The effect of time on venom replacement was determined by a paired T-test. ANOVA was performed to analyze differences in venom weight between fed and unfed treatments. RESULTS: Nutritional suspension was found to have a significant effect on the quantity of venom produced. SDS-PAGE gel and FPLC revealed that the components of the venom collected from both groups were similar, indicating that four weeks is an adequate time to regenerate key venom components but not replenish initial venom quantities. CONCLUSIONS: Venom production was found to be affected by starvation.

Anti-inflammatory effect of Natterins, the major toxins from the Thalassophryne nattereri fish venom is dependent on TLR4/MyD88/PI3K signaling pathway.

Here we evaluated whether Natterins affect the leukocyte-endothelial cell interaction, hampering leukocyte mobilization and extravasation. Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle using intravital microscopy. We reported that low doses of Natterins interfere with the cell capturing, inhibiting the interaction of blood neutrophils with the post-capillary venules induced by the TLR4 agonist LPS, or the chemokine KC. Using endotoxemic mice challenged with LPS, we confirmed that Natterins reduce neutrophil accumulation in the peritoneum exudates. The rolling of leukocytes induced by KC or LPS was not impaired in Natterins-treated TLR2, MyD88 deficient or TLR4 mutant mice, indicating that TLR2- or TLR4-MyD88-mediated signals are required for the anti-inflammatory effect of Natterins. The inhibitory effect was not influenced by endogenous regulators of inflammation such as IL-10, corticosteroids, the HO-1 or the antagonist of the receptor of IL-1, nor by the disruption of their proteolytic activity. However, it was completely dependent on the activation of serine/threonine phosphatases and the PI3K signaling pathway, but independent on increased proteasome activity. This work started asking how the main toxins in the T nattereri venom contributes for the deficient influx of inflammatory leukocytes, which consequently drive to the delayed inflammatory reaction finalization in injured tissue; and finished demonstrating that Natterins can control the leukocyte-endothelial wall interactions in a mechanism dependent on negative signals derived from TLR2-TLR4/Myd88 signaling cascade. Interestingly, we confirmed that the antagonist effect of Natterins is mediated by the activation of serine/threonine phosphatases and by the key signaling PI3K molecule.

DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples.

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells.

Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15µg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.

Studies on pharmacological properties of mucus and sting venom of Potamotrygon cf. henlei.

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout the rivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posing a serious public health problem even if not properly reported. The accidents usually involve fishermen and bathers, and to date there is no effective treatment for the injured. Considering these facts and limitations of studies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describe the principal pharmacological and certain biochemical properties of the mucus and sting venom. We found that mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Our results also indicate that the inflammatory cellular influx observed could be triggered by the venom and mucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested for antimicrobial activity in which only the mucus presented activity. It could be inferred from the present study that P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically active components.

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii.

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.

Determination of tetrodotoxin and its analogs in the puffer fish Takifugu oblongus from Bangladesh by hydrophilic interaction chromatography and mass-spectrometric detection.

Tetrodotoxin (TTX) and its analogs (TTXs), widely distributed among marine as well as terrestrial animals, induce dangerous intoxications. These highly potential toxins are also known as the causative agent of puffer fish poisoning. A newly developed highly sensitive method for determination of TTXs based on hydrophilic interaction chromatography and mass-spectrometric detection is presented. TTX, anhydrotetrodotoxin, 11-deoxytetrodotoxin and trideoxytetrodotoxin were determined in separated tissues of Bangladeshi marine puffers, Takifugu oblongus. TTX was predominant in skin, muscle and liver, whereas trideoxytetrodotoxin preponderated in the ovary. The toxicity of the various tissues was determined by a mouse bioassay.

Ostreopsis sp., a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons.

A clone of toxic dinoflagellate Ostreopsis sp. and six specimens of a parrotfish Scarus ovifrons were collected in October 1997 at Tokushima Prefecture, Japan. Ostreopsis sp. was cultured in ESM medium for 16 days, and after rearing the cell pellet (about 4.0x10(5) cells) was extracted with 50% methanol, partitioned between an aqueous layer and 1-butanol layer, and biochemically tested. Similarly, the crude toxin from S. ovifrons was extracted, and tested. The mice injected with each 1-butanol layer from Ostreopsis sp. and S. ovifrons showed the common symptoms of convulsion, drowsiness and collapse, and died within 48 h. The lethal potency of Ostreopsis sp. was calculated to be 1.0x10(-4) MU/cell. All specimens of S. ovifrons were found to be toxic, where the highest potency was determined as 2 MU/g in muscle of one specimen. After being injected with toxins, the serum creatine phosphokinase levels of mice were found to be elevated. Toxins from Ostreopsis sp. and S. ovifrons showed delayed haemolytic activity with mouse and human erythrocytes, which was inhibited by an anti-palytoxin (PTX) antibody antibody and ouabain. Toxins from Ostreopsis sp. and S. ovifrons thus resembled each other, and strongly suggested to be PTX or its akin substance. Additionally, a considerable number of adherent Ostreopsis sp. was found in the gut contents of S. ovifrons during the heavy occurrence of Ostreopsis sp. in October 1997 at Tokushima Prefecture. From the above results, it can be strongly postulated that the dinoflagellate Ostreopsis sp. is the origin of PTX which is sequestered by the parrotfish S. ovifrons through food chain.

[Neurological manifestations of fish, mollusk and shellfish poisoning]. / Manifestations neurologiques des intoxications par les poissons, mollusques et crustacés.

Sea-food poisoning is observed in several areas of the world. Intoxication results from ingestion of fresh fish, mollusks, or shellfish contaminated by toxins produced by microorganisms (dinoflagellates). Neurological manifestations are sometimes associated with signs and may be life-threatening. We describe here the principle toxins, their geographic distribution, clinical manifestations, therapeutic management, and possible prevention measures.

Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry.

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M+H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M+H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M+H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels <50% and were subjected to large losses of activity on purification indicating that unstable ciguatoxins were present. A possible conversion of C-CTX-1 into C-CTX-1a was identified when flesh was cooked, without changes in toxicity. In conclusion, HPLC/MS characterised 12 C-CTXs accumulated by C. latus at variable levels.

Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning.

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for Hinfl was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for Hinfl was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.

[Recent advances in tetrodotoxin research].

One century has passed since fugu toxin was named tetrodotoxin (TTX) by Tahara. Chemical problems such as crystallization of tetrodotoxin and subsequent structure determination were solved by research groups headed by Tsuda, Hirata, Woodward, and Mosher. The International Symposium on the Chemistry of Natural Products in Kyoto (1964) was well known as symposium which the structure of TTX was internationally clarified. Since the first isolation of toxin from taricha torosa (imori) as natural source except for fugu fishes, distribution of toxin in nature has been widely investigated. And, it was proved that toxin is not produced by fugu fishes, but rather is formed by sea bacteria (30 sp.) such as Alteromonas sp, Vibrio sp, Shewanella. However, it seems to be difficult to explain the tetrodotoxin accumulation at high concentration in fugu by only toxin production by bacteria. TTX analogues were isolated from natural origins such as crabs, fish, annelids, and algae. Based on the structure of these toxin analogues, the biosynthesis of toxin and the structure-activity relationship (Na+ channel) were proposed by Yasumoto-Yamashita. The findings of wide distribution of toxin in nature may be attributed to development of highly sensitive detection method for toxin. The interesting proposal for the biosynthesis and the structure activity, and the detection method for toxin are outlined in this review.

Haff disease: from the Baltic Sea to the U.S. shore.

Haff disease, identified in Europe in 1924, is unexplained rhabdomyolysis in a person who ate fish in the 24 hours before onset of illness. We describe a series of six U.S. patients from 1997 and report new epidemiologic and etiologic aspects. Although Haff disease is traditionally an epidemic foodborne illness, these six cases occurred in two clusters and as one sporadic case.

Neutralization of Thalassophryne nattereri (niquim) fish venom by an experimental antivenom.

T. nattereri (niquim) is a venomous fish involved in many human accidents in Brazil. The clinical picture includes mild local erythema, severe edema, intense pain and rapid progression to necrosis. The present therapy with anti-inflammatory and analgesic drugs is ineffective and, therefore, we decided to assess serum therapy as an alternative treatment using an experimental antivenom. The antivenom used was raised in rabbits showing an ELISA antibody titer of 1:8,192,000 and its ability to neutralize lethality, necrosis, nociception and edema was evaluated both by pre-incubating the venom with antivenom before injection into mice or by independent injections of venom and antivenom. Lethality was completely neutralized by pre-incubation (ED(50)=141.5 microl/mg) while necrosis and nociception were neutralized by pre-incubation or the independent injection of antivenom. Edema was only partially prevented even when large amounts of antivenom were used. These data suggest that antivenom may be a promising treatment for patients stung by T. nattereri and suggest the viability of producing a horse antivenom for use in clinical trials.

Toxicities and distribution of tetrodotoxin in the tissues of puffer fish found in the coast of the Baja California Peninsula, Mexico.

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).

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus.

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6-11 x higher hemolytic activity and 10x higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic alpha-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3-4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic alpha-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Cs 1.

Determination of Na channel blockers in paralytic shellfish toxins and pufferfish toxins with a tissue biosensor.

The biosensor consisted of a sodium electrode and covered with the frog bladder membrane within a flow cell was tested for the estimation of tetrodotoxin (TTX) and saxitoxin (STX). This sensor was applied to detect very low amounts of the Na+ channel blockers, STX and TTX, in different shellfishes and swellfishes. A good agreement was obtained between TTX activities determined by mouse assay and amounts of Na+ channel blockers estimated by frog membrane sensor. The lowest level of TTX (fg) that can be determined by frog membrane sensor does not cause human poisoning. The channel blockers in short-necked clam, which was assumed to be STX, were monitored by this sensor continuously every week for one year. It was discovered that the STX content increased from July until September and then decreased from October until March. The biosensor proposed here may be used for the estimation of STX and TTX conventionally in the future.

Isolation and identification of novel protein kinase genes from the round-spotted pufferfish (Tetraodon fluviatilis) genomic DNA.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish, Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-one T. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identified pk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selected pk genes in a variety of tissues. We found that erbB3, pku a, mrk, CaMK I, CaMKIIgamma, and two novel kinase genes (133 and 3-26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.

Occurrence of paralytic shellfish poisons in Thai freshwater puffers.

Screening tests were carried out on the toxicity of freshwater puffers Tetraodon leiurus complex and Tetraodon suvatii collected from Udonthani province, north-eastern Thailand. Toxicity was highest in the liver and varied according to the location and season of fish catch. Fish which were reared in tap water for 3 months reduced the toxicity substantially. Partial purification was achieved by an ultrafiltration technique. Toxin components were consequently identified by high-performance liquid chromatography. It was found that toxins separated from the eggs, liver, skin and muscle of these puffers were composed of saxitoxin, neosaxitoxin and decarbamoylsaxitoxin.