Search

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lipophilic phycotoxins (LPTs) and domoic acid (DA) in Antarctic seawater, as well as parts of the South Pacific and the Southern Indian Oceans were systematically investigated. DA and six LPTs, namely pectenotoxin-2 (PTX2), okadaic acid (OA), yessotoxin (YTX), homo-yessotoxin (h-YTX), 13-desmethyl spirolide C (SPX1), and gymnodimine (GYM), were detected. PTX2, as the dominant LPTs, was widely distributed in seawater surrounding Antarctica, whereas OA, YTX, and h-YTX were irregularly distributed across the region. The total concentration of LPTs in surface seawater ranged from 0.10 to 13.57 ng/L (mean = 2.20 ng/L). ∑LPT levels were relatively higher in the eastern sea areas of Antarctica than in the western sea areas. PTX2 was the main LPT in the vertical profiles, and the PTX2 concentration was significantly higher in the epipelagic zone than water depths below 200 m. The predominant sources of PTX2 and OA in Antarctic sea areas are likely to be Dinophysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The effects of yessotoxins (YTXs) produced by the dinoflagellate Protoceratium reticulatum in the early stages of bivalves have not been studied in detail. The present study evaluates the effects of P. reticulatum and YTXs on the survival and feed ingestion of veliger larvae of Argopecten purpuratus. Larvae were 96 h-exposed to 500, 1000 and 2000 P. reticulatum cells mL -1 , and their equivalent YTX extract was prepared in methanol. Results show a survival mean of 82 % at the highest density of dinoflagellate, and 38 % for larvae with the highest amount of YTX extract. Feed ingestion is reduced in the dinoflagellate exposure treatments as a function of cell density. Therefore, the effect of YTXs on A. purpuratus represents a new and important area of study for investigations into the deleterious effects of these toxins in the early stages of the life cycle of this and, potentially, other bivalves., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The dinoflagellates Protoceratium reticulatum and Lingulodinium polyedra are potential yessotoxin (YTX) producers, which have been associated with blooms responsible for economic, social, and ecological impacts around the world. They occur in Iberian waters, but in this region, little is known of their ecophysiology and toxin profiles. This study investigated the growth and toxin production of two strains of each species, from the Portuguese coast, at 15 °C, 19 °C, and 23 °C. Growth curves showed higher growth rates at 19 °C, for both species. YTX and three analogs (homo YTX; 45-OH YTX; 45-OH homo YTX) were investigated by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), and the presence of other analogs was investigated by Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS). No evidence of toxin production was found in L. polyedra. By contrast, YTX and 45,55-diOH-YTX were detected in both strains of P. reticulatum. These results confirm P. reticulatum as a source of yessotoxins along the Portuguese coast and add to the observed high intraspecific variability on YTX production of both species, at a global scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The distribution characteristics of lipophilic marine biotoxins (LMTs), such as yessotoxins (YTXs) and pectenotoxins (PTXs) in phytoplankton, mussels, and commercial seafood were determined for the southern coast of South Korea. Gonyaulax spinifera and Dinophysis acuminata, which are the causative microalgae of YTXs and PTXs, were recorded during summer. Homo-YTX and PTX-2 were predominantly detected in phytoplankton (max: 5.7 μg g -1 ww), whereas only YTXs were detected in mussels (max: 1.1 μg g -1 ww). LMT concentrations in mussels were positively correlated with those in phytoplankton. However, there was a 1-month time gap in maximum LMT concentrations between mussels and phytoplankton. Homo-YTX was detected in commercial seafood, including red scallop and comb pen shell. However, homo-YTX concentrations in shellfish were below the recommended value of the European Food Safety Authority (3.75 mg YTX equivalents kg -1 ); thus, the consumption of this seafood was not considered to be a significant risk for human health., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Florida red tide is a natural phenomenon caused by the dinoflagellate, Karenia brevis. Karenia brevis blooms produce potent toxins (brevetoxins) that can cause neurotoxic and respiratory illness in humans and marine life. Red tides were recorded by Spanish explorers as early as the 17 th century, however published red tide studies before 1940 are unavailable. Recent studies have suggested that red tide events may be becoming more frequent, intense, and longer lasting, which may be linked to modern land development and changing water quality. While the scientific record of modern red tides is relatively short, the distributions and concentrations of chemical biomarkers (e.g., brevetoxins produced by K. brevis) in coastal-marine sediments can potentially be used to study historic red tides. This study aims to quantify the concentration and vertical distribution of brevetoxins in coastal Southwest Florida (SWFL) sediment cores in order to determine if downcore brevetoxins may potentially be used to reconstruct historic red tide events. Sediment samples were radiometrically dated using 210 Pb and subsamples were analyzed utilizing liquid chromatography/triple quadrupole mass spectrometry (LC-MS/MS) for brevetoxin congeners, namely, PbTx-1, PbTx-2, PbTx-3, and PbTx-5. The 210 Pb-dated sediment cores represent ∼60-80 years of brevetoxin accumulation and total brevetoxin (ΣPbTx) concentrations in sediment cores varied from below detection limits to 25.3 ng g  -   1 of dry sediments. Highest concentrations were found in surficial sediments (top 0-3 cm) and may indicate brevetoxin preservation from the 2017-2019 red-tide event. The down-core preservation and variability of brevetoxin indicate its potential use as a chemical biomarker to assess long-term red tide intensities and frequencies. This research is a first step towards reconstructing historic red tide events from sedimentary chemical biomarkers and may allow for future assessment of the human impacts on red tide frequency, intensity and duration., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The spectroscopic ellipsometry (SE), and attenuated internal reflection spectroscopic ellipsometry (TIRE) are promising methods in label-free biosensing applications. An ellipsometer running under surface plasmon resonance (SPR) conditions has unique advantages over other SPR-based methods in terms of sensitivity and real-time/label-free measurement capability. In this study, both SE and TIRE-based brevetoxin B (BTX) sensors were developed using two anti-BTX aptamers reported before. A new aptamer sequence was also derived from these two antiBTX aptamers using predictive modeling tools and an exclusion method. All three antiBTX aptamers' analytical performances were quite competitive in terms of both detecting range and detection limits. However, the selectivity of the previously reported aptamers against analogs of BTX was poor at low detection ranges, especially for okadaic acid. Furthermore, the selectivity of the derived aptamer was lower than its predecessors. The sensors were capable of detecting BTX in the range of 0.05 nM-1600 nM in the TIRE and 0.5 nM-2000 nM in the SE configuration. The detection limits of the sensors were 1.48 nM (1.32 ng/mL) and 0.80 nM (0.72 ng/mL) for SE and TIRE configurations, respectively. Both configurations have been used successfully to detect BTX standards spiked into real fish and shrimp samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Brevetoxins (PbTx) are a well-recognized group of neurotoxins associated with harmful algal blooms, and specifically recurrent "Florida Red Tides," in marine waters that are linked to impacts on both human and ecosystem health including well-documented "fish kills" and marine mammal mortalities in affected coastal waters. Understanding mechanisms and pathways of PbTx toxicity enables identification of relevant biomarkers to better understand these environmental impacts, and improve monitoring efforts, in relation to this toxin. Toward a systems-level understanding of toxicity, and identification of potential biomarkers, high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was utilized for metabolic profiling of zebrafish (Danio rerio) embryos, as an established toxicological model, exposed to PbTx-2 (the most common congener in marine waters). Metabolomics studies were, furthermore, complemented by an assessment of the toxicity of PbTx-2 in embryonic stages of zebrafish and mahi-mahi (Coryphaena hippurus), the latter representing an ecologically and geographically relevant marine species of fish, which identified acute embryotoxicity at environmentally relevant (i.e., parts-per-billion) concentrations in both species. HRMAS NMR analysis of intact zebrafish embryos exposed to sub-lethal concentrations of PbTx-2 afforded well-resolved spectra, and in turn, identification of 38 metabolites of which 28 were found to be significantly altered, relative to controls. Metabolites altered by PbTx-2 exposure specifically included those associated with (1) neuronal excitotoxicity, as well as associated neural homeostasis, and (2) interrelated pathways of carbohydrate and energy metabolism. Metabolomics studies, thereby, enabled a systems-level model of PbTx toxicity which integrated multiple metabolic, molecular and cellular pathways, in relation to environmentally relevant concentrations of the toxin, providing insight to not only targets and mechanisms, but potential biomarkers pertinent to environmental risk assessment and monitoring strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Brevetoxins (BTX) are pharmacologically active, lipid soluble cyclic polyether neurotoxins that are known to cause a wide range of neurological symptoms in humans.Harvesting and consumption of infected molluscs provide an entry point for BTXs into, the food chain, causing long-term health effects on accumulation for individuals, commonly in people with a compromised immune system and existing allergies. This study is an acoustic assay that has been constructed using a 9 MHz AT-cut quartz crystal resonator modified by attaching a specific single-stranded DNA aptamer. The DNA oligo modifies its conformation to attach itself to the binding site of the incoming BTX molecule resulting in a change in frequency on the QCR. A small Δf value was observed for lower concentrations of BTX indicating a small change in mass deposited on the crystal surface, while the opposite was true for higher concentrations. Cross-species behavior was evaluated using samples of similar origin, molecular weight and a combination of two toxins. The LOD of the fabricated QCR is 220 nM which is lower than the maximum recommended residue limit in food samples. Fresh mussel samples were spiked with known concentrations of BTX to evaluate its sensitivity in a food matrix. No interaction with other compounds was observed. Overall, this sensor finds potential application in the food sector (fishing units) where mussels are tested and graded for allergens and toxins before reaching the customer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (Na V ) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target Na V . The sensitivity and specificity of the assay to compounds activating the Na V are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60-80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3-2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay., (Copyright © 2021. Published by Elsevier B.V.)

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC-MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45-OH homo-YTX were detected throughout the monitored period. 45-OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45-OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The Benguela Current in the Atlantic is one of the four major upwelling systems on the Eastern boundary of the world ocean. Thus the coastal regions off Namibia are prone to high primary productivity that can lead to Harmful Algae Blooms as this nutrient rich water reaches the euphotic zone. Yessotoxins (YTXs) produced by G. spinifera were detected in Namibian phytoplankton field samples in 2011. Isolation of G. spinifera cultures from this location in 2012 enabled molecular genetics work and further liquid chromatography-mass spectrometry assessment of toxin profiles. The molecular work grouped the Benguela G. spinifera with other toxic G. spinifera strains originating from Italy and New Zealand. The main YTX analogs present in the Benguela G. spinifera are homo-YTX, YTX and a hydroxylated analogue. This work adds important knowledge on the occurrence of Harmful Algae Blooms in this region and is of relevance for safety., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparède & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Effects of Ptychodiscus brevis toxin (PbTx) analogs on the spinal synaptic transmission in neonatal rats in vitro were evaluated. PbTx1/PbTx2 had aromatic groups and PbTx3/PbTx4 had aliphatic groups. All the analogs depressed monosynaptic reflex (MSR) and polysynaptic reflex (PSR) in a concentration-dependent manner. The maximal depression of MSR (75% from initial) and PSR (96%) was at 84 microM for PbTx1. Concentration to produce 25% inhibition from initial (IC25) by PbTx1 for MSR and PSR was < or =2.8 microM. The maximal depression of MSR (80%) was at 96 microM and PSR (100%) was at 32 microM by PbTx2. IC25 for MSR and PSR were 5.5 microM and <3.2 microM, respectively. PbTx3 decreased MSR by 25% maximally (=IC25) at 36 microM. The depression of PSR fluctuated and was maximal (75%) at 108 microM and IC25 was 6.2 microM. PbTx4 depressed MSR and PSR at the maximum of 35% at 32 microM and IC25 for MSR was 8.3 microM and for PSR was 35 microM. Rank order of potency of toxins for depressing MSR was PbTx1>PbTx2>>PbTx4>PbTx3; and for PSR it was PbTx2>PbTx1>PbTx3>>PbTx4. Results indicate that the toxins having aromatic groups exhibited greater neurotoxicity.

Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH(2)OC(=O)-2, 6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified mu- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease., (Copyright 2000 Academic Press.)

Background: The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na(+) channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists., Results: PbTx-3 and benzoyl-PbTx-3 elicited Na(+) channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na(+) channel openings. alpha-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na(+) channels that were not exposed to PbTx-3. beta-Naphthoyl-PbTx-3 reduced openings of Na(+) channels that were not exposed to PbTx-3., Conclusions: Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin.

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.

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., (Copyright 2000 Academic Press.)

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment., (Copyright 1999 Academic Press.)

Brevetoxins (PbTx) are a family of marine polyether toxins that exert their toxic action by activating voltage-sensitive sodium channels. Two forms of brevetoxin, PbTx-2 and -3, induce hepatic cytochrome P4501A1, measured as ethoxyresorufin O-deethylase (EROD) activity, in redfish and striped bass. P4501A1 induction is transcriptionally regulated through the binding of a ligand, typically a planar aromatic compound, to the aryl hydrocarbon receptor (AhR) and subsequent complex formation with the dioxin response element (DRE), an upstream regulatory region of the CYP1A1 gene. To determine if PbTx, a nonaromatic compound, induced EROD by this mechanism, two sets of experiments were performed. Initially, saturation binding assays with PbTx-2, -3, and -6 were carried out to determine if PbTx-2, -3, or -6 was an AhR ligand. Results showed that PbTx-6 inhibited specific binding of dioxin to the AhR, whereas PbTx-2 and -3 had no effect. Subsequently, gel retardation assays showed that PbTx-6 caused a concentration-dependent increase in AhR-DRE complex formation. The most abundant and neurotoxic forms of brevetoxin, PbTx-2 and -3, did not appear to be involved in this process. However, PbTx-6, the epoxide which is a likely biotransformation product, is at least one of the forms of PbTx involved in EROD induction.

Maitotoxin (MTX), a water-soluble polyether obtained from the marine dinoflagellate Gambierdiscus toxicus increased intracellular calcium in a concentration-dependent manner in fibroblasts obtained from human skin. The effect of this toxin was both saturable and of high affinity, showing an apparent half activation constant of 450 fM. The toxin did not release intracellular calcium storage compartments nor did the release of these compartments with thapsigargin or ionomycin affect the toxin response. The toxin effect was reduced significantly by pre-incubating the cells with 0.1% trypsin for 30 min, strongly suggesting that the toxin receptor is a plasmalemmal protein. The effect of MTX was partially inhibited by diphenoxylate.

Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.

Maitotoxin (MTX) is a highly potent marine toxin that activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane. This results in calcium overload that rapidly leads to cell death. We now report that maitotoxin (0.1-1 nM) induces calpain activation in both SH-SY5Y neuroblastoma cells and fetal rat cerebrocortical cultures. MTX-induced calpain activation was confirmed by the presence of autolytic fragmentation of both subunits of calpain. Secondly, the formation of calpain-produced alpha-spectrin breakdown products (150 and 145 kDa) was observed. We were also able to detect intracellular hydrolysis of a peptide substrate (succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin) by activated calpain in MTX-treated cells. Calpain inhibitors (calpain inhibitor I, MDL28170 and PD150606) inhibited spectrin breakdown and SLLVY-AMC hydrolysis in MTX-treated SY5Y cells. Our results suggest that (i) calpain is activated as a result of the maitotoxin-induced calcium influx; and (ii) coupling with the in situ calpain assays, maitotoxin would be a useful tool in investigating the physiologic and pathophysiologic roles of calpain in neuronal cells.

Background: Brevetoxins, involved in the 'red tide' as well as shellfish poisoning, are known to bind to cell membranes and membrane proteins. Brevetoxin B (BTX-B) interacts specifically with neuronal sodium channels. We recently found that BTX also induces selective ion movements across lipid bilayers through transmembrane BTX self-assemblies., Results: We examined the self-assembly of several BTX derivatives in the presence and absence of cations and lipid bilayers using the powerful porphyrin chromophores as circular dichroism labels. BTX derivatives self-assemble into tubes, which can bind to metals both when soluble and when inserted into the bilayer to form transmembrane pores. Depending on the tendency of the BTX derivative to self-aggregate (the critical 'micelle' concentration, cmc), it may aggregate in solution before membrane insertion, or may insert itself into the membrane as a monomer before assembling the pore., Conclusions: The active BTX-B complex in lipid bilayers is a cyclic, transmembrane self-assembly consisting of antiparallel aligned BTX molecules that can mediate selective ion movement through membranes. The differences in pore formation mechanisms between BTX derivatives may be reflected in differences in pore formation by natural BTX variants, perhaps explaining their varying levels of toxicity.

Background: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules., Results: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states., Conclusions: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.

In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.

Stimulation of m1 and of m3 muscarinic receptors has previously been shown to increase intracellular cAMP levels in a variety of cells. Although the mechanism underlying this response is not fully understood, it has been hypothesized to be secondary to the IP3-mediated rise in intracellular calcium. In order to determine whether other means of elevating intracellular calcium also raise cAMP levels, we stimulated SK-N-SH human neuroblastoma cells with bradykinin or with maitotoxin. Both of these agents stimulated phospholipase C, stimulated inositol phosphate release and elevated cAMP levels, thus demonstrating that this cAMP response is not unique to muscarinic receptor stimulation.

Eutrophication phenomena in marine coastal waters can today be explained on the basis of natural or anthropogenic causes. Undesirable effects and also sanitary problems in both types of eutrophication are often produced, but they may differ greatly in frequency and significance. Some algal biotoxins can affect both marine animals and man, whilst others affect man alone. From data currently available it appears that the sanitary state of man can be affected through the digestive, respiratory and cutaneous apparatus. Four main dinoflagellate biointoxications are now recognized: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhoetic shellfish poisoning (DSP), and venerupin poisoning. Other biointoxications are due to a diatom bloom responsible for amnesic shellfish poisoning (ASP) and to blue algae blooms which have effects on the skin and the respiratory tract. All these marine toxins are considered and particular attention is paid to: producing organisms, chemistry of the components, compromised sea foods, methods of analysis, occurrence worldwide, human intoxications, toxicology and mechanism of action on a molecular level, therapeutical notes, tolerance levels and remarks on safety. Attention is also paid to the relationship between the anthropogenic eutrophication and PSP and DSP since these are the most widespread biointoxications from toxic marine dinoflagellates in the world today and for which the European Economic Community (EEC) is proposing health legislation such as tolerance limits and methods for official analysis. In view of the harmful potential of coastal anthropogenic eutrophication, the main current committment of various countries concerns control. Finally, it is important to develop a suitable monitor research system using all the specific standards of allowed toxic substances, and also research on effective antiodotes against all biotoxins.

The penetration and distribution of [3H]PbTx-3 into pig skin was determined using in vivo and in vitro methods. The dose used in each topical study was 0.3-0.4 micrograms/cm2 skin, with dimethylsulfoxide as the vehicle. In the in vivo study, mean cutaneous absorption after 48 h (expressed as percentage of the dose) was 11.5% (n = 3). In the in vitro study, mean cutaneous absorption after 48 h was 1.6% (n = 12), when based on accumulation of radioactivity in receptor fluid, or 9.9% when based on receptor fluid and dermis. [3H]PbTx-3 readily penetrated through the epidermis into the dermis, reaching maximal dermal accumulation at 4 h (9.1% in vivo and 18% in vitro). At 24 h, the amount in the dermis decreased to 2.3% and 15% in vivo and in vitro, respectively and at 48 h the amount in the dermis decreased to 8.2% in vitro. These results demonstrate the important role of the dermis as a reservoir for a lipophilic compound in both in vivo and in vitro percutaneous absorption studies.

The permeability coefficients (Kp) for tritium-labelled water (THO) were determined in human and monkey skin, and monkey buccal mucosa. Kp of human skin (0.47 x 10(-3) cm/h) correlated favorably with previous reports. Kp of hydrated monkey skin for THO (0.77 x 10(-3) cm/h) was not significantly different (P greater than 0.05) from Kp of hydrated human skin (0.88 x 10(-3) cm/h). Kp of monkey buccal mucosa for THO (6.15 x 10(-3) cm/h) was significantly greater than that for monkey skin. Penetration and disposition of [3H]PbTx-3 into layers of monkey buccal mucosa and skin was determined. [3H]PbTx-3 (5-7 microCi) dissolved in 2 ml of water was applied to epithelial/epidermal surface (2.8 cm2) at zero time. The relative percent dose recovered from the upper layers of buccal mucosa (epithelium) and skin (epidermis) varied, but at each time interval was less than 2.5% of the dose. At most of the time intervals (2-24 h), a larger percent of the dose was recovered from the inner layer of the buccal mucosa (lamina propria) than from the inner layer of skin (dermis). After 24 h, as much as 34 or 13% of the dose was recovered from lamina propria or dermis, respectively. At each time interval studied, less than 2% of dose of [3H]PbTx-3 penetrated into the receptor fluid which bathed the inner surfaces of the lamina propria or dermis. The results of this study demonstrate that monkey buccal mucosa is more permeable than skin to THO and PbTx-3.

The MMQ pituitary cell line, which expresses a membranal dopamine receptor, was used to examine the individual contributions of dopamine and protein kinase C (PKC) to control of the intracellular calcium concentration. The calcium concentrations, monitored with the fluorescent dye Indo-1, increased in response to elevated K+, BAY K8644, and maitotoxin, implicating the presence of voltage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 60 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8644 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent D-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8644 or maitotoxin.

Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANF-mediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.

In the present study we used 235-1 cells, a prolactin secreting clone derived from a pituitary tumor. In these cells maitotoxin, a calcium channels activator, likely acting on voltage sensitive calcium channels, increases intracellular free calcium measured by Quin 2 technique. Maitotoxin stimulation of calcium flux was inhibited both by nicardipine and verapamil in a dose dependent manner. Pertussis toxin pretreatment does not modify maitotoxin activation of calcium channels, while completely abolishes nicardipine inhibition of maitotoxin induced voltage sensitive calcium channels activation, without affecting verapamil effect. These results suggest a possible involvement of a pertussis toxin sensitive G protein in dihydropyridine inhibition of voltage sensitive calcium channels.

In the present study we investigated the effect of the interleukin 1 beta on intracellular free calcium concentrations in 235-1 cell line both in basal conditions and after stimulation by the calcium channel activator maitotoxin. Interleukin 1 beta (from 0.01 pM to 10 nM) was unable to significantly affect basal cytosolic free calcium levels in acute conditions. The preincubation of these cells with interleukin 1 beta for 48h modulates maitotoxin stimulation of calcium fluxes without modifying basal intracellular free calcium levels. Low concentrations of interleukin 1 beta (0.01 pM, 1 pM) caused a marked reduction of intracellular free calcium concentrations increase induced by maitotoxin while higher doses of the monokine potentiated maitotoxin stimulation of calcium fluxes. The specificity of interleukin 1 beta effect was tested by means of polyclonal anti-interleukin 1 beta antibody (titer 1:100) which significantly abolished the inhibitory effect of interleukin 1 beta on free cytosolic calcium levels. These results show that a long lasting interaction of interleukin 1 beta with its receptor is able to influence voltage-sensitive calcium channels activation induced by maitotoxin in 235-1 cells.

Maitotoxin (MTX) provoked a dose-dependent increase in both 45Ca efflux and insulin release from rat pancreatic islets perifused in the presence or absence of glucose, provided that Ca2+ was present in the perifusate. The stimulatory effect of MTX on 45Ca outflow was enhanced by CGP 28392. The toxin did not reduce 86Rb outflow and 86Rb inflow. It is suggested that the secretory response to MTX is mediated by direct activation of voltage-dependent Ca2+ channels.

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.