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PubMed2002

Characterization of F-actin depolymerization as a major toxic event induced by pectenotoxin-6 in neuroblastoma cells.

Pectenotoxins are a group of marine toxins produced by dinoflagellates and formerly included within the group of diarrhetic shellfish poison or toxins (DSP or DST) because of their physico-chemical properties. However, toxicological data on pectenotoxins are still very scarce and its mechanism of action is largely unknown, but toxicity in laboratory animals has been demonstrated by intraperitoneal injection. In this report, we present results of in vitro toxicological assessment of pectenotoxin-6, a derivative of the parental toxin pectenotoxin-2 first isolated from toxic scallops. Results obtained demonstrate an specific time- and dose-dependent depolymerization of F-actin in neuroblastoma cells exposed to pectenotoxin-6 (half-maximal effect about 700 nM at 24 hr). The change in the state of polymerization of actin was not accompanied by other major effects on specific signal transduction pathways or cell survival rate. Pectenotoxin-6 does not modify cytosolic calcium levels either in a calcium containing or calcium-free medium in human lymphocytes. Only when capacitative calcium influx was first activated, the toxin addition significantly decreased the following calcium influx. In these cells, pectenotoxin-6 only modifies cAMP (adenosine 3',5'-cyclic monophosphate) levels in calcium-free conditions. In addition, no effect on cell attachment or apoptosis induction was observed at micromolar concentrations of pectenotoxin-6. Therefore, we conclude that cytoskeletal disruption is a key mechanism of PTX6-induced toxicity in eukaryotic cells.

Actins↗

PubMed1978

A purification of Helix pomatia alpha amylase involving a novel affinity-binding procedure.

Shellfish glycogen was cross-linked by treatement with cyanogen bromide followed by 1,6-diaminohexane. The resulting, insoluble product efficiently adsorbed Helix pomatia alpha amylase [(1 linked to 4)-alpha-D-glucan glucanohydrolase] from crude solutions of the enzyme at 0 degrees, but only poorly at higher temperatures. A method was developed for the purification of Helix pomatia alpha amylase involving formation of an enzyme-adsorbent complex in the cold and recovery of the alpha amylase by suspending the washed complex in buffer at 37 degrees. After chromatography of the desorbed alpha amylase on a column of Bio-Gel P-60, the enzyme was homogenous as judged by poly(acrylamide)-gel electrophoresis. An overall purification of 360-fold was achieved with a recovery of 35%.

Adsorption↗

PubMed2004

Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response.

Microcystins, potent heptapeptide hepatotoxins produced by certain bloom-forming cyanobacteria, are strong protein phosphatase inhibitors. They covalently bind the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), thereby influencing regulation of cellular protein phosphorylation. The paralytic shellfish poison, okadaic acid, is also a potent inhibitor of these PPs. Inhibition of PP1 and PP2A has a dualistic effect on cells exposed to okadaic acid or microcystin-LR, with both apoptosis and increased cellular proliferation being reported. This review summarises the existing data on the molecular effects of microcystin-LR inhibition of PP1 and PP2A both in vivo and in vitro, and where possible, compares this to the action of okadaic acid.

Animals↗

PubMed2003

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis).

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Animals↗

PubMed2002

Direct detection of yessotoxin and its analogues by liquid chromatography coupled with electrospray ion trap mass spectrometry.

A liquid chromatography mass spectrometry (LC-MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC-MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995-1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported.

Animals↗

PubMed2000

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production.

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.

Journal Article↗

PubMed2003

A model of fish preference and mortality under hypoxic water in the coastal environment.

Eutrophication and associated occurrence of hypoxic condition could cause significant damage to marine ecosystems, resulting in considerable economic losses to fisheries and aquaculture and is a major source of stress that fish often have to contend with in order to survive. This problem is likely to be exacerbated in the coming years, since the wastewater treatment facilities is unlikely to catch up with increasing human activities. Moreover, large-scale reclamation projects in coastal areas have recently been increased, and these activities certainly have adverse impacts on water quality and fisheries resources. Coastal construction has a significant role in the development of hypoxic water by changing the current and mixing pattern of water. Changes in species composition and decreases in species richness and diversity have been well documented in hypoxic systems. Hypoxia could cause endocrine disruption in fish and eliminate populations of sensitive species. Shallow coastal areas are of great importance for the special nursery of fish and shellfish and land reclamation in these areas cause strong damage to fisheries. Although the tolerance of aquatic life to hypoxia is known, there is no information about the mortality of fish caused by hypoxia because fish can swim around it and no modeling study has yet been carried out. Criteria that influence the movement of fish are: amounts of food, water temperature and depth, dissolved oxygen concentration and nature of seabed. However, among these, water temperature and dissolved oxygen are the most crucial parameters that affect survival, movement and growth of fish. In this paper, a model of fish preference and mortality for environmental conditions was developed and applied to the Hakata Bay where hypoxic water occurs every summer. For the purpose of this study, a field survey of fish behavior under hypoxic water was conducted by releasing marbled sale (Pleuronectes yokohamae) in the inner bay. Moreover, a series of preference tests for DO, salinity and temperature in the laboratory were conducted in order to decide preference parameters of fish. Using the results of both field and laboratory studies, a sub-module of fish preference and mortality was coded within an integrated hydrothermal and eutrophication model (CHEM) to predict the behavior and mortality of marbled sale when hypoxia would occur in the bay. The model could reasonably simulate the behavior of the fish under hypoxia. An assessment of the impact of the ongoing land reclamation project of about 401 ha in the Wajiro tidal flat zones at the head of the bay on the fisheries resources was also conducted using the model. The results showed that the artificial land lowered the mortality rate of fish under hypoxic condition in the bay during the summer period.

Animals↗

PubMed2003

Assay of phosphatase activity and ATP biomass in tideland sediments and classification of the intertidal area using chemical values.

Detailed studies of the phosphatase activity in the tidelands were examined including the localization of the phosphatase activities and the spatial distributions of the activity in the tidelands around Hiroshima Bay, the Aki-Bingo Open Sea and Isahaya Bay of Kyushu on the western side of Japan. The results indicate that the phosphatase activity of the sediment fraction accounts for 98% of the tideland activity. Furthermore, we classified 19 tidelands into four groups by a cluster analysis using two parameters: phosphatase activities and ATP (adenosine 5(')-triphosphate) biomass. The first group is a member of the tidelands located in areas remote from the cities. They have a low phosphatase activity and low ATP biomass and the seawater in the tideland is very clean. The second is a group with a low phosphatase activity and high ATP biomass. In the tidelands belonging to this group, large numbers of benthos and shellfish can be observed. The third group has a high phosphatase activity and a relatively low ATP biomass. The estuary tidelands located near Hiroshima City belong to the third group. The last one is tidelands with a high phosphatase activity and high ATP biomass. In this group, the ratio of the phosphatase activity to ATP biomass is relatively low. In the summer, we observed deterioration in the water quality and accumulation of sludge on the sea bottom in the tideland of the last group.Moreover, we employed a multiple regression analysis and proposed an equation to obtain the group number of the cluster analysis using the assayed values of the phosphatase activity and the ATP biomass.

Adenosine Triphosphate↗

PubMed2003

Impacts of the Nakhodka heavy-oil spill on an intertidal ecosystem: an approach to impact evaluation using geographical information system.

A major heavy-oil spill from the Russian tanker Nakhodka occurred in the Sea of Japan on 2 January 1997. We investigated the impacts of this spill on a rocky intertidal ecosystem along the southern coast of the Sea of Japan. We selected Imago-Ura Cove as our study site to observe temporal changes along the oiled shore, because minimal cleaning effort was made in this area. Field surveys were conducted every autumn and spring from 1997 to 2000. We measured coverage by macroalgae in 1 x 1-m(2) quadrats and counted the animals in 5 x 5-m(2) quadrats along the intertidal zone. Changes in the ecosystem caused by the oil spill were analyzed by applying a geographical information system (GIS) to the Sea of Japan for the first time. The GIS showed that following the accident there were heavily oiled areas in sheltered regions, but these decreased over the three years. It also showed that coverage by macroalgae and the number of animals increased, although some species of algae with microscopic sporophyte generations, and some populations of perennial shellfish, remained stable or decreased during the study period. GIS was able to trace temporal changes in intertidal communities resulting from the impacts of heavy oil on flora and fauna at a spatial scale of 10-100 m. GIS is thus a practical tool for visualizing, analyzing, and monitoring changes in an ecosystem polluted by oil, taking into account topographic differences along the coastline.

Accidents↗

PubMed2003

Stable carbon and nitrogen isotope signatures indicate recovery of marine biota from sewage pollution at Moa Point, New Zealand.

Stable carbon and nitrogen isotopes have been used to assess sewage contamination of a sewage outfall, discharging milli-screened effluent into Moa Point Bay, New Zealand, and monitor the recovery of flora and fauna after the outfall's closure. An initial study characterising the extent of the discharge and the effects on seaweed (Ulva lactuca L.), blue mussels (Mytilus galloprovincialis) and limpets (Cellana denticulata) from the area, showed effects of the sewage discharge on flora and fauna were localised within in the bay. The immediate area surrounding the discharge area was found to contain limited biodiversity, with an abundance of Ulva lactuca, a bright green lettuce-like seaweed, typically found in areas with high nutrient input, limpets and small blue mussels. The nitrogen isotopic signature (delta15N) is shown to be a good tracer of sewage pollution in seaweed and associated grazers (i.e. limpets) as a result of the increased contribution of urea and ammonia to seawater nitrogen derived from the effluent. The carbon isotopic signature (delta13C) is suggested as a more appropriate sewage tracer for mussels, which filter feed the effluent's particulate organic matter from the water. Lower carbon:nitrogen ratios were found in Ulva lactuca sampled from around the outfall region compared to uncontaminated control sites. However carbon:nitrogen ratios do not vary significantly amongst shellfish species. After closure, monitoring continued for 9 months and showed that the carbon and nitrogen isotopic signatures of algae (Ulva lactuca L.) returned to similar control site levels within 3 months. Limpet and blue mussels (Cellana denticulata and Mytilus galloprovincialis) showed slower recovery times than the Ulva lactuca, with detectable levels of the sewage-derived carbon and nitrogen remaining in the animal's tissue for up to 9 months.

Animals↗

PubMed2003

Application of trichloroacetic acid (TCA) to extraction of soft body for the determination of tissue Cd, Cu, Pb and Zn in the prosobranch Hydrobia ulvae (Pennant).

The application of trichloroacetic acid (TCA) as a shell extractant for preparation of soft body parts with reference to tissue metal concentrations (Cd, Cu, Pb, Zn) in shellfish has been evaluated on the example of the mud snail Hydrobia ulvae, a small marine prosobranch densely present in rocky and soft-bottom habitats of the eastern Atlantic. A solution of 0.1 M TCA was tested on individuals treated according to two different protocols: (1) thawed after freezing ("non-dried") and (2) thawed and air-dried to a constant weight ("dried"). Two points were investigated in detail to improve the method: individual soft tissue dry weight and tissue metal concentration following a standard digestion method. In both instances, the results were compared with those from manually dissected snails. Conditions for total shell decalcification of 60 individuals (3-4 mm long) were 5.5 h in 20 ml of 0.1 M TCA.No differences in individual soft tissue weight were observed between the treatments, indicating good efficiency of the TCA extraction with respect to weight of soft body parts. In contrast, tissue metal concentrations varied among treatments. The TCA extraction of the dried animals had a good recovery for Cd, most likely due to the lower solubility of Cd vital cellular components (proteins and mineral concretions) from the dried tissue. Satisfactory recoveries of the tissue concentrations of Cu and Pb were obtained for the non-dried individuals. This might be related to the specific distribution of metals in the organism (namely in the digestive glands and gonads) and their different chemical reactivity with TCA after the tissue was dried. Limited susceptibility of Zn-bearing protein bindings to complexing with TCA also accounts for significantly lower concentrations of Zn in the mud snail's soft tissue that was extracted. The 0.1 M TCA solution is therefore recommended for extraction of the shells of Hydrobia ulvae for tissue determination of Cd, Cu and Pb; however the treatment protocol does affect metal recovery and thus a consistent procedure should be followed. The extracted metals from the soft tissues and shells of the mud snails (on the basis of both metal concentrations and contents) were ranked in order of increasing contribution of soft body parts to the total (shell+tissue): Pb<Cd<Zn<Cu.

Animals↗

PubMed1998

The analysis of DNA adduct formation, removal and persistence in the common mussel Mytilus edulis exposed to 4-nitroquinoline 1-oxide.

32P-postlabelling was used for the detailed analysis of 4-nitroquinoline 1-oxide (4-NQO) induced DNA adduct formation, removal and persistence in the marine shellfish Mytilus spp. The results had a number of important implications concerning the use of such DNA adducts as dosimeters of environmental genotoxin exposures. Our studies indicated that the maintenance of the Mytilus specimens under controlled laboratory conditions can result in the induction of 'stress-related adducts' seemingly related to the nature of the experimental set-up. The studies also indicated that the absorption and activation of genotoxins in this species appear to affect the rate of adduct formation, and that the maximum levels of adducts may not necessarily be induced immediately after the cessation of a genotoxin exposure. In addition, Mytilus specimens were shown to possess a significant capacity to remove these genotoxin-induced DNA adducts. The removal of these adducts appeared to be biphasic in nature, with the rapid removal of a large proportion of adducts occurring within 48 h of the cessation of the exposure, followed by a slow rate of adduct removal over the remaining period of the studies. Despite the relatively efficient removal of the majority of these genotoxin-induced DNA adducts, a proportion remained up to 56 days after the initial exposure. The persistence of these genotoxin-DNA adducts, combined with the information on the rates of adduct removal, indicated that under well-defined conditions, such adducts could serve as suitable biomarkers of environmental contamination.

4-Nitroquinoline-1-oxide↗

PubMed1995

Outbreak of Vibrio cholerae 01 in Hong Kong related to contaminated fish tank water.

An outbreak of 12 cholera cases, caused by Vibrio cholerae eltor inaba, occurred in Hong Kong during a three week period in June-July 1994. Only adults of both sexes were affected. Epidemiological investigations showed linkage in all cases with consumption of seafood, including shellfish, mantis shrimps and crabs. Microbiological findings demonstrated that contaminated seawater in fish tanks used for keeping alive these seafoods is the most likely vehicle of transmission. Aggressive control measures, promptly instituted, included prohibition of use of contaminated typhoon shelter water in fish tanks, use of seawater with E. coli counts below 610 organisms/100 ml, and the banning of unlicensed food sampans in typhoon shelters. These measures, coupled with public announcements and an active health education campaign on food safety and personal hygiene, abruptly terminated the outbreak. Places which practise the use of seawater, from probable contaminated sources, to keep alive their seafood for human consumption should be alerted to the possibility of transmission of Vibrio cholerae through this route.

Adolescent↗

PubMedDate not supplied

Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.

Amphibian Proteins↗

PubMed2001

Possible source of tetrodotoxin in the starfish Astropecten scoparius.

The seasonal variations of toxicity and stomach contents in toxic starfish Astropecten scoparius were detected. The average highest specimen toxicity, expressed as tetrodotoxin (TTX), was 16,821 mouse units (MU). The toxin was composed of TTX only, except April's sample containing mainly TTX along with minor paralytic shellfish poisons. The composition in the stomach of less and more toxic starfish was mainly Veremolpa scabra and Umborium suturale, respectively. The toxicities of U. suturale and V. scabra, found in the starfish stomach were 65 and 33 MU, respectively. For those collected from the coastal waters, U. suturale was toxic with average toxicity value of 77 MU/g, but V. scabra was nontoxic. The toxin in the specimens of U. suturale collected from either the digestive gland of starfish or the coastal waters, was TTX and anhydroTTX only. It indicates that the starfish A. scoparius might mainly accumulate high amount of TTX from U. suturale. Furthermore, both small gastropods U. suturale and Natica psuestes are first reported to contain TTX.

Animals↗

PubMed2001

Delayed haemolytic activity by the freshwater puffer Tetraodon sp. toxin.

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

Acrylamides↗

PubMed2001

The occurrence of 7-epi-pectenotoxin-2 seco acid in the coastal waters of the central Adriatic (Kastela Bay).

Okadaic acid (OA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA) were identified as the toxic determinants in mussels from the central Adriatic Sea. The nature of the pectenotoxin-2 derivative was confirmed by chromatographic comparison with toxins present in algae extracts of Dinophysis acuta from Ireland, and by mass spectrometric analysis. The origin of shellfish toxicity has been associated with the occurrence of the Dinophysis species. This is the first report on the incidence of 7-epi-PTX-2SA in the Adriatic region.

Animals↗

PubMed2001

Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium.

Minute amount of Brevetoxin PbTx-3 (400 microg; 0.446 micromol) was converted into an hemisuccinate derivative (PbTx-3 HS) then covalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) in a reversed micellar medium. According to the efficient cyclic synthetic procedure described, the epitope density of the conjugates was around 10 and 20 for OVA and BSA carriers, respectively. The kinetics of antibody production in sequential sera harvested from a single BALB/c mouse immunised by multiple intraperitoneal (i.p.) injections of PbTx-3-BSA conjugate was performed by enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies (MAbs) against PbTx-3 were selected from fusion of the mouse immune splenocytes with the P3-X63-Ag 8.653 myeloma cells. In competitive inhibition ELISA experiments, both polyclonal antibodies and MAbs exhibited strong cross-reactivity (> or = 100%) to other PbTx-2-type toxins (PbTx-2 and -9) but low or moderate cross-reactivity (6-15%) to a PbTx-1-type toxin (PbTx-1). Moreover, using these two MAbs, a low cross-reactivity with okadaic acid (3%) was noticed but no significant cross-reactivity was observed with two ciguatoxins (CTX-1B and CTX-3C) over the concentration range studied. The apparent dissociation constant (K(D)) for the interaction of these MAbs with free PbTx-2-type toxins was in the 10(-6)-10(-7)M range. The performance of this MAb-based assay (limit of detection approximately 5ng/well; working range=8-150ng/well) coupled with adequate extraction methods would provide an alternative assay to the mouse i.p. bioassay for routine shellfish monitoring. This production and characterisation of MAbs using small amount of polyether toxins in a reversed micellar medium appear most valuable for the development of immunoassays to other highly potent but poorly available marine polyether toxins like ciguatoxins (CTXs).

Animals↗