Unique toxin profile of a Mediterranean Ostreopsis cf. ovata strain: HR LC-MS(n) characterization of ovatoxin-f, a new palytoxin congener

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.     

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.     

First episode of shellfish contamination by palytoxin-like compounds from Ostreopsis species (Aegean Sea, Greece).

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.     

Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas

Massive blooms of the benthic dinoflagellate Ostreopsis ovata Fukuyo have recently occurred along the whole Italian coastlines, both Tyrrhenian and Adriatic, resulting sometimes in benthonic biocenosis sufferings and, occasionally, in human health problems. In this work, two strains of O. ovata collected in 2006 along the Adriatic and Tyrrhenian coastlines and grown in culture were studied to characterize their growth and toxin profile. The two strains showed different cell volumes, the Adriatic strain being nearly twice bigger than the Tyrrhenian, but they had similar slow growth rates. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that both strains produce putative palytoxin (pPLTX) and ovatoxin-a (OVTX-a), a palytoxin-like compound presenting 2 oxygen atoms less than palytoxin.Toxin content was determined at the end of the stationary and exponential growth phases and reached the highest value in the Adriatic strain at the end of the stationary phase, with concentrations of 353.3 μg l^?1 for OVTX-a and 30.4 μg l^?1 for pPLTX. Toxin released in the growth medium was also measured and resulted to be the highest at the end of the stationary phase, suggesting that a long lasting bloom could enhance the toxin content in the water and cause toxic effects in people inhaling the aerosol.     

Isolation of Prorocentrum lima (Syn. Exuviaella lima) and diarrhetic shellfish poisoning (DSP) risk assessment in the Gulf of California, Mexico.

A benthic toxic dinoflagellate identified as Prorocentrum lima (Syn. Exuviaella lima), and designated as strain PRL-1, was isolated from the coast of El Pardito (Coyote) Island in Baja California Sur, Mexico, after a fisherman poisoning incident involving consumption of liver from Lutjanus colorado, and Mycteroperca prionura fish. Purification and culturing was done in ES-Si medium, under 12:12 light/dark cycle (4 x 20 W cool-white fluorescent lamps), at 22 degrees C and constant stirring during 28 days. Whole cells were toxic to Artemia franciscana and its methanolic extract to mouse and to the marine yeast Debaryomyces hansenii. Chromatographic analysis (TLC and HPLC-MS) of such extract indicated an unusual proportion (1:2) okadaic acid (OA) and dinophysistoxin-1 (DTX-1). Estimated total toxin content by mouse bioassay (based on OA toxicity) was 19 pg/cell, a value significantly higher than that found by HPLC-MS (about 5.2 pg/cell, taking into account OA and DTX-1 only), suggesting that additional toxic components of unidentified nature are detected with the bioassay. This is the first report of a successful isolation and culturing of a toxic dinoflagellate from the Gulf of California, Mexico.     

A review on the effects of environmental conditions on growth and toxin production of Ostreopsis ovata

Since the end of the 1990s the occurrence of blooms of the benthic dinoflagellates Ostreopsis spp. is spreading in many tropical and temperate regions worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. Ostreopsis ovata has been found to produce palytoxin-like compounds, a class of highly potent toxins. As general, the highest abundances of Ostreopsis spp. are recorded during warmer periods characterized by high temperature, salinity, and water column stability. Moreover, as these cells are easily resuspended in the water column, the role of hydrodynamism in the blooms development and decline has been highlighted. The environmental conditions appear, therefore, to be one of the main factors determining the proliferation of these species as testified by several field surveys. Laboratory studies on the effect of environmental parameters on growth and toxicity of O. ovata are rather scarce. With regard to the effects of temperature, culture results indicate that different strains blooming along Italian coasts displayed different optima, in accordance to blooming periods, and that higher toxin levels correlated with best growth conditions. Additionally, in relation to an Adriatic strain, cell growth positively correlated with the increase in salinity, while toxicity was lowest at the highest salinity value (i.e. 40). For the same strain, both nitrogen and phosphorus limitation determined a decrease in cell toxicity showing different behaviour with respect to many other toxic dinoflagellates.     

Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity

Attempts were made to isolate the bacteria from toxic dinoflagellate Alexandrium minutum T1 and to study the effect of these bacteria on the growth and toxicity of A. minutum T1. It was found that intracellular bacterial species including Pasteurella haemolytica, Pseudomonas vesicularis, and Sphingomonas sp., and extracellular bacterial species including Pasteurolla pneumotropica, Morganella wisconsensis, Flavobacterium oryzihabitans, Pseudomonas pseudomallei, and Sphingomonas sp. All of them were cultured and determined to have non-PSP-producing ability. The maximum cell number of A. minutum cultured without isolated bacteria was higher than that cultured with isolated bacteria. The total toxicity of A. minutum cultured with bacteria was similar to that of A. minutum T1 cultured without bacteria from lag phase to stationary phase, but it was lower after stationary phase. The growth of A. minutum T1 cultured without antibiotics was also better than that cultured with antibiotics. The total toxicity of A. minutum cultured without antibiotics was higher than that of A. minutum cultured with antibiotics. However, the cell toxicity of A. minutum did not decrease even if the culture medium was added with antibiotics.     

Effects on adhesiveness and hydrophobicity of sub-inhibitory concentrations of netilmicin

The effect of sub-inhibitory concentrations (SICs) of netilmicin on bacterial hydrophobicity and adhesiveness to conjunctival cells was investigated. One strain each of Pseudomonas aeruginosa, Pseudomonas spp., Staphylococcus aureus and S. epidermidis was investigated for its susceptibility to netilmicin, its adherence to conjunctival cells and to the effect of hydrocarbon hexadecane before and after treatment with SIC of netilmicin. All of the bacteria tested were susceptible to netilmicin except for Pseudomonas spp. which showed intermediate resistance. Netilmicin-treated Pseudomonas strains exhibited a lower level of hydrophobicity towards n-hexadecane compared with non-treated strains, while netilmicin-treated S. epidermidis and S. aureus showed a slight increase of hydrophobicity. Adherence of the two Pseudomonas strains to conjunctival cells was significantly reduced after growth in the presence of netilmicin, while the adherence of the two staphylococci was only slightly reduced.     

Evidence for strain-specific differences in benzene toxicity as a function of host target cell susceptibility

It has long been recognized that benzene exposure produces disparate toxic responses among different species or even among different strains within the same species. There is ample evidence that species- or strain-dependent differences in metabolic activity correlate with the disparate responses to benzene. However, bone marrow cells (the putative targets of benzene toxicity) may also exhibit species- or strain-dependent differences in susceptibility to the toxic effects of benzene. To investigate this hypothesis, two sets of companion experiments were performed. First, two strains of mice, Swiss Webster (SW) and C57B1/6J (C57), were exposed to 300 ppm benzene via inhalation and the effects of the exposures were determined on bone marrow cellularity and the development of bone marrow CFU-e (Colony Forming Unit-erythroid, an early red cell progenitor). Second, bone marrow cells from the same strains were exposed in vitro to five known benzene metabolites (1,4 benzoquinone, catechol, hydroquinone, muconic acid, and phenol) individually and in binary combinations. Benzene exposure, in vivo, reduced bone marrow cellularity and the development of CFU-e in both strains; however, reductions in both these endpoints were more severe in the SW strain. When bone marrow cells from the two strains were exposed in vitro to the five benzene metabolites individually, benzoquinone, hydroquinone, and catechol reduced the numbers of CFU-e in both strains in dose-dependent responses, phenol weakly reduced the numbers of the C57 CFU-e only and in a non-dose-dependent manner, and muconic acid was without effect on cells from either strain. Only benzoquinone and hydroquinone exhibited differential responses to CFU-e from the two strains and both of these metabolites were more toxic to SW cells than to C57 cells. Six of the ten possible binary mixtures of metabolites were differentially toxic to the CFU-e from the two strains and five of these mixtures were more toxic to SW cells than to C57 cells. Thus, SW mice were more susceptible to the toxic effects of inhaled benzene and their bone marrow cells were more severely affected by in vitro exposure to benzene metabolites. The binary combinations containing phenol produced little or no enhancement of the toxic effects of the non-phenol metabolites. The weak toxic response induced by phenol, whether delivered alone or in binary mixtures, suggests that little metabolism occurred during the 48 h of the in vitro exposures since benzoquinone and hydroquinone, which were clearly toxic when added to the CFU-e culture system, are formed by further metabolic oxidation of phenol. Thus, strain-dependent differential metabolism appeared to play a minimal role in the disparate toxicity observed in the in vitro studies, implying that the diverse responses were due to inherent differences in the susceptibilities of the CFU-e to the toxic action of the benzene metabolites.     

Toxicity to medaka fish embryo development of okadaic acid and crude extracts of Prorocentrum dinoflagellates

Chronic and subchronic toxicity following exposure to the DSP (Diarrhetic shellfish poisoning) toxin okadaic acid (OA) is receiving increasing attention as a public human health biohazard. However information on ecological impacts induced by proliferation of the OA producing dinoflagellate Prorocentrum is scarce. In order to analyse the toxicity of these substances, in vivo experiments were conducted on medaka fish (Oryzias latipes) embryos used as an experimental model. The study was focused on two strains of benthic Prorocentrum species, P. arenarium and P. emarginatum, naturally found in the Indian Ocean. Sample extracts (crude extracts, CE) were obtained from algal cultures and their toxic potential was explored. Their OA (and derivatives) content was evaluated by two methods: one based on chemical analysis using HPLC-MS, the other based on screening the inhibiting effect on protein phosphatase PP2A. P. arenarium extracts inhibit PP2A and the active toxin was confirmed as being OA by HPLC-MS. In contrast, P. emarginatum showed negative results regardless of the method used. The development of medaka fish embryos kept in medium containing pure OA or Prorocentrum CE was examined. Survival rates were reduced up to 100% depending on the concentrations used of both OA and CE of P. arenarium, while no effect was observed with CE of P. emarginatum. Anatomopathological studies of surviving embryos indicate that OA treatment resulted in significant increases in liver and digestive tract areas compared to controls. P. arenarium treated surviving embryos exhibited significant quantitative increases of global body and vitellus areas. Together, our results indicate that the toxic effects to medaka embryos development of pure OA and P. arenarium extracts containing OA are distinguishable. The differences may indicate the presence of additional toxic substance(s) (or molecules able to modulate OA impact) in the P. arenarium CE that probably are not present in P. emarginatum.     

Remarkable difference in accumulation of paralytic shellfish poisoning toxins among bivalve species exposed to Pyrodinium bahamense var. compressum bloom in Masinloc Bay, Philippines.

Seasonal variation of bivalve toxicity was monitored in association with the abundance of the toxic dinoflagellate Pyrodinium bahamense var. compressum in Masinloc Bay, Luzon Island. Among 7 species of bivalve, 6 species became toxic during a bloom of the dinoflagellate. However, remarkable difference in the toxicity was observed among the species. The toxicity of thorny oyster Spondylus squamosus was the highest among the species, showing more than 30 times that of safety consumption level after the peak bloom of the dinoflagellate, while other bivalve species showed much lower toxicity. The toxicity of thorny oyster decreased under absence of the dinoflagellate, but this species maintained a considerably high toxicity throughout a year. Similar trend was observed in penshell Atrina vexillum in a small scale, indicating that these species in the bay are not safe for human consumption almost throughout a year. The toxicity of green mussel Perna viridis increased to significant level during a bloom, but it decreased rapidly when the dinoflagellate disappeared. Toxin analysis of cultured and natural cells showed typical toxin profile of the dinoflagellate. Toxin profile of all the bivalve species reflected the characteristic toxin profile of the dinoflagellate.     

Latitudinal Variation in the Toxicity and Sexual Compatibility of Alexandrium catenella Strains from Southern Chile

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5–55° S) 50 years ago and is responsible for most of the area’s cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell⁻¹), with the more toxic strains occurring in the north (−40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000–1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.     

Temperature-dependent sensitivity of growth and photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains of Microcystis aeruginosa to the herbicide atrazine

The temperature-dependent sensitivities of two algal species and two strains of cyanobacteria to the photosynthesis-inhibiting herbicide atrazine were evaluated in order to understand how the interaction between acclimation temperature and herbicide will affect growth and photosynthesis of aquatic microorganisms. The green alga Scenedesmus obliquus, the diatom Navicula pelliculosa and a toxic and non-toxic strain of Microcystis aeruginosa were acclimated to three different temperatures (10, 15 and 25°C) and exposed to five concentrations of the herbicide atrazine (0-0.15μM) for 72h. Growth, photosynthetic yields, energy fluxes within photosystem II and pigment content were then measured as potential responses to each treatment. With the exception of N. pelliculosa, the toxicity of atrazine was higher when microorganisms were acclimated to lower temperatures. N. pelliculosa was not only the most tolerant to atrazine, but also had a similar sensitivity to this herbicide at every temperature. The observed differences in growth sensitivity to atrazine at low temperature are associated with the ability of algae and cyanobacteria to cope with high excitation pressure, by increasing its protective carotenoid content and non-photochemical energy dissipation. Our results demonstrate that future guidelines for the protection of aquatic life should consider water temperature as an important factor influencing the toxicity of atrazine to aquatic microorganisms.     

Chemical speciation and toxicity of metals assessed by three bioluminescence-based assays using marine organisms

Metal toxicity is a function of the biology of the target organism and the chemical speciation of the metal. The toxicity of 11 metals was assessed with three cell-based bioassays based on marine organisms: the bacterium Photobacterium phosphoreum of the Microtox bioassay, an environmental strain of P. phosphoreum, and photocytes isolated from the brittlestar Ophiopsila californica. Metal speciation was calculated for three commonly used media: NaCl-based Microtox bioassay medium, artificial seawater glycerol, and artificial seawater. Decreased bioluminescence was considered a proxy for cell toxicity. In all three assays the elements Cd and Hg exhibited similar speciation as well as similar toxicity profiles. The element Cu was toxic in all three assays despite different metal speciation for the P. phosphoreum bioassay. The element Ag was toxic to both bacterial strains but not to photocytes despite a similar chemical speciation for all three assays. In general, the Microtox bioassay was sensitive to all metals (except Pb), whereas the photocytes were the least sensitive to the metals. The heightened response of the Microtox bioassay probably resulted from a combination of the limited complexing power of the medium and the greater sensitivity of the bacterial strain.     

Effects of cyanobacterial lipopolysaccharides from microcystis on glutathione-based detoxification pathways in the zebrafish (Danio rerio) embryo

Cyanobacteria ("blue-green algae") are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems.     

Effects of temperature on the growth and carbohydrate production of three benthic dinoflagellate species from Hainan Island,South China Sea

Ecotoxicology - Dinoflagellates in the genera Ostreopsis and Gambierdiscus are typical members of the marine benthic community particularly in tropical areas. Their geographic distribution has...     

Toxicity evaluation and microbiota response of the lined sole Achirus lineatus (Chordata: Achiridae) exposed to the light petroleum water-accommodated fraction (WAF)

ABSTRACT

Exposure to contaminants might directly affect organisms and alter their associated microbiota. The objective of the present study was to determine the impact of the petroleum-water-accommodated fraction (WAF) from a light crude oil (API gravity 35) on a benthic fish species native from the Gulf of Mexico (GoM). Ten adults of Achirus lineatus (Linnaeus, 1758) were exposed to a sublethal WAF/water solution of 50% v/v for 48 hr. Multiple endpoints were measured including tissue damage, presence of polycyclic aromatic hydrocarbons (PAHs) metabolites in bile and gut microbiota analyses. Atrophy and fatty degeneration were observed in livers. Nodules and inflammation were detected in spleen, and structural disintegration and atrophy in the kidney. In gills hyperplasia, aneurysm, and gills lamellar fusion were observed. PAHs metabolites concentrations in bile were significantly higher in exposed organisms. Gut microbiome taxonomic analysis showed significant shifts in bacterial structure and composition following WAF exposure. Data indicate that exposure to WAF produced toxic effects in adults of A. lineatus, as evidenced by histological alterations and dysbiosis, which might represent an impairment to long-term subsistence of exposed aquatic organisms.     

Equilibrium partitioning as the basis for an integrated laboratory and field assessment of the impacts of DDT,DDE and DDD in sediments

Many of the most biologically productive portions of streams are backwater areas which support large populations of benthic macroinvertebrates. The sediments in these locations and their associated macroinvertebrate communities are frequently subjected to chemical inputs and physical perturbations. Historically, assessment of the effects of contaminants in sediments have emphasized chemical analyses and either laboratory toxicity tests or in-stream monitoring of benthic macroinvertebrate community structure. However, combining the chemical and biological approaches provides a more powerful assessment technique. Such an integrated approach, combining laboratory water-only and sediment toxicity tests with Hyalella azteca and Chironomus tentans, field surveys of benthic macroinvertebrate community structure and evaluation of chemical data using equilibrium partitioning theory was used to assess the effects of DDT, DDE and DDD (collectively termed DDTR) in the sediments of the Huntsville Spring Branch-- Indian Creek (HSB--IC) stream system in the southeastern USA. Benthic macroinvertebrate populations in the HSB--IC system still appear to be adversely affected by DDTR residues within the sediments even though DDT discharges to the stream were stopped over 20 years ago and a major remediation project was completed in the late 1980s. This conclusion is based on a weight of evidence approach which incorporates (1) the observed sediment toxicity to C. tentans and H. azteca in laboratory tests, (2) the identification of DDTR as the likely cause of effects observed during laboratory toxicity tests, (3) the absence of appropriate sensitive species from groups such as the Ephemeroptera, Plecoptera, Trichoptera and Amphipoda, (4) the presence of reduced numbers of both total individuals and species of chironomids and oligochaetes relative to nearby streams not contaminated by DDTR and (5) the observed distribution of benthic macroinvertebrates in relation to organic carbon-normalized concentrations of DDTR and equilibrium partitioning-based predicted sediment toxic units of DDTR     

Effects of toxic extracts and purified borbotoxins from Prorocentrum borbonicum (Dinophyceae) on vertebrate neuromuscular junctions.

Benthic dinoflagellates of the genus Prorocentrum are common in tropical and subtropical water and several species produce phycotoxins potentially involved in human toxic outbreaks. The toxic dinoflagellate Prorocentrum borbonicum collected at La Réunion Island (France) was cultured in laboratory. A crude extract of the organism displayed significant toxicity in mice characterized by progressive limb paralysis, severe dyspnea, and death, and the toxicity was retained, after partition, in the extract's butanol-soluble fraction (BSF). Electrophysiological experiments characterizing the fraction's effect on isolated vertebrate neuromuscular preparations revealed that it depolarizes the muscle membrane and reduces the driving force for endplate potentials (EPPs) evoked by nerve stimulation, blocking directly- and indirectly-elicited muscle twitches. The depolarization induced by P. borbonicum BSF was not due to Na(+) influx through voltage-dependent Na(+) channels, since tetrodotoxin neither prevented nor suppressed the depolarization. However, ouabain, a specific ligand of the Na/K ATPase, reduced the depolarization. These results suggest the presence of palytoxin-like compounds in the fraction. HPLC-MS and MS/MS analysis showed the presence of several toxins having identical UV absorbance, among which two new isomeric toxins, borbotoxin-A and -B, of molecular mass of 1037.6 Da were isolated. The purified borbotoxin-A, had no effect on the resting membrane potential of muscle fibers and did not affect directly-elicited muscle twitches. However, the toxin reduced nerve-evoked muscle twitches, in a dose-dependent manner, reduced EPPs' amplitudes and completely blocked miniature endplate potentials. These observations suggest that the main action of borbotoxin-A is to block post-synaptic nicotinic ACh receptors.     

Toxicological studies on palytoxin and ostreocin-D administered to mice by three different routes

Palytoxin (PLT) first isolated from zoanthids is extremely lethal to animals by intraperitoneal or intravenous administration but shows little toxicity by gavage dosing in contradiction to the occurrence of fatal poisoning due to PLT-containing seafood.In order to fully elucidate its potential risks to human we evaluated the toxicological effects via three ways of dosing: gavage, intra-tracheal administration (IT) and sublingual administration. A new analog, 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin isolated from the dinoflagellate Ostreopsis siamensis and named ostreocin-D (OSD), was also used for comparison, additionally conducted by i.p. By gavage dosing, both toxins did not produce death in mice at the maximum dosage of 200 μg/kg of PLT and 300 μg/kg of OSD. Addition of dietary lipid components to PLT solutions for gavage or use of ulcerated mice did not alter the results, indicating no enhancement of PLT absorption. The two toxins were most toxic by the IT route, causing bleeding and alveolar destruction in the lung and resultant death at 2 μg/kg of PLT, and 11 μg/kg of OSD. Both toxins also induced organ injuries after 24 h when dosed by sublingual administration at about 200 μg/kg. The injuries became fatal when PLT was dosed 2 or 3 times. The results pointed to the necessity of taking multiple approaches to assess the potential health risks due to PLT and its analogs in food and environments.