Special Issue "Marine Toxins Detection"

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Marine and Freshwater Toxins".

Deadline for manuscript submissions: closed (31 July 2019).

Special Issue Editor

Prof. Takeshi Yasumoto
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Guest Editor
Japan Food Research Laboratories, Tama Laboratory, 6-11-10 Nagayama, Tama City, Tokyo 206-0025, Japan

Special Issue Information

Dear Colleagues,

Marine toxins have diverse faces. They threaten human health, damage environments, undermine fisheries and aquaculture, and obstruct the trade of fisheries products. On the other hand, some toxins are highly esteemed as unique research tools to probe biological and biochemical phenomena, for example, tetrodotoxin and saxitoxin, which are used to probe voltage sensitive sodium ion channels, or okadaic acid, which is used as a potent and specific inhibitor of protein phosphatase 2A. The geographical occurrence of marine toxins is widening, the producing organisms becoming diverse, with new structures and functions surfacing. A wide variety of methods are developed in pursuit of high sensitivity, specificity, rapidity, and practicality. This Special Issue aims to showcase the latest advances in detection methods developed based on their structures and function. LC–MS analysis is now available using previously unavailable standards for ciguatoxins and metabolite brevetoxins. A successful collaboration between synthetic chemists and immunochemists to produce sandwich ELISA for the detection of ciguatoxins will be presented.

Prof. Takeshi Yasumoto
Guest Editor

Manuscript Submission Information

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Keywords

  • ciguatera toxins
  • palytoxins
  • ichthyotoxins
  • tetrodotoxin
  • cyclic imines
  • cyclic polyethers
  • LC–MS
  • ELISA/functional assay

Published Papers (11 papers)

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Research

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Article
Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications
Toxins 2019, 11(12), 722; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11120722 - 11 Dec 2019
Cited by 15 | Viewed by 1382
Abstract
In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence [...] Read more.
In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g−1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure–activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay
Toxins 2019, 11(11), 632; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11110632 - 31 Oct 2019
Cited by 8 | Viewed by 1161
Abstract
Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. [...] Read more.
Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94–0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Chemiluminescent Receptor Binding Assay for Ciguatoxins and Brevetoxins Using Acridinium Brevetoxin-B2
Toxins 2019, 11(10), 580; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11100580 - 09 Oct 2019
Viewed by 1167
Abstract
Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely differ in structures depending on their geographic origins, which range from the Pacific Ocean and the Indian Ocean to the [...] Read more.
Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely differ in structures depending on their geographic origins, which range from the Pacific Ocean and the Indian Ocean to the Caribbean Sea. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of bivalve shellfish contaminated with brevetoxins (BTXs). Structurally, both CTXs and BTXs consist of fused ether rings aligned in a ladder shape. Pharmacologically, they bind at the same site (site-5) of voltage-gated sodium channels. However, the great structural diversity and the rare availability of reference toxins hinder LC-MS and ELISA methods, which operate on structure-based recognition. In this study, we prepared a chemiluminescent ligand, acridinium BTXB2 (ABTX), and tested its suitability for use in competitive binding assays to detect CTXs and BTXs. The affinity of ABTX to the rat brain synaptosome estimated by Ki (1.66 pM) was approximately two-fold higher than that of PbTx-3 (BTX3). In addition, the equilibrium dissociation constant (KD) was 0.84 nM, the maximum number of binding was 6.76 pmol toxin/mg protein, and the detection limit was 1.4 amol. The assays performed on samples spiked with CTX3C or BTXB4 (N-palmitoylBTXB2) at 0.2–1.0 ng CTX/g fish flesh, and 200–800 ng BTXB4/g shellfish showed a linear relationship between the theoretical and observed toxin amounts. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Development of Ultra-Performance Liquid Chromatography with Post-Column Fluorescent Derivatization for the Rapid Detection of Saxitoxin Analogues and Analysis of Bivalve Monitoring Samples
Toxins 2019, 11(10), 573; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11100573 - 01 Oct 2019
Cited by 5 | Viewed by 1409
Abstract
Saxitoxin (STX) and its analogues produced by toxic dinoflagellates accumulate in bivalves, and routine monitoring of bivalves is important to prevent cases of human poisoning. In this study, we describe a rapid detection method for the analysis of STXs using ultra-performance liquid chromatography [...] Read more.
Saxitoxin (STX) and its analogues produced by toxic dinoflagellates accumulate in bivalves, and routine monitoring of bivalves is important to prevent cases of human poisoning. In this study, we describe a rapid detection method for the analysis of STXs using ultra-performance liquid chromatography with post-column fluorescent detection and to investigate water depths and sampling points optimal for shellfish toxin monitoring. Cultured scallops (Mizuhopecten yessoensis) and mussels (Mytilus galloprovincialis) collected from various water depths and sampling points were used in this study. Irrespective of bivalve species, toxin concentrations in bivalves were lower at deeper water depths. The toxin concentrations of bivalves did not differ greatly when bivalves were collected from the same bay. Although the levels of contamination of bivalves with STXs can depend on various environmental and geographical factors, our findings are useful for formulating a sampling protocol for the prevention of harvesting contaminated shellfish. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin
Toxins 2019, 11(10), 551; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11100551 - 20 Sep 2019
Cited by 3 | Viewed by 1200
Abstract
A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production [...] Read more.
A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04)
Toxins 2019, 11(9), 539; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11090539 - 16 Sep 2019
Cited by 5 | Viewed by 1656
Abstract
Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle [...] Read more.
Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12β-deoxygonyautoxin 3 (12β-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC–MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan
Toxins 2019, 11(9), 521; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11090521 - 09 Sep 2019
Cited by 2 | Viewed by 1549
Abstract
Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography–mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) [...] Read more.
Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography–mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Article
Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels
Toxins 2019, 11(9), 513; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11090513 - 03 Sep 2019
Cited by 4 | Viewed by 1537
Abstract
Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials [...] Read more.
Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2–3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Communication
Oscillatoxin I: A New Aplysiatoxin Derivative, from a Marine Cyanobacterium
Toxins 2019, 11(6), 366; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11060366 - 21 Jun 2019
Cited by 10 | Viewed by 1547
Abstract
Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various [...] Read more.
Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various aplysiatoxin derivatives from a M. producens sample obtained from the Okinawan coastal area. The frozen sample was extracted with organic solvents. The ethyl acetate layer was obtained from the crude extracts via liquid–liquid partitioning, then separated by HPLC using a reversed-phase column. Finally, 1.1 mg of the compound was isolated. The chemical structure of the isolated compound was elucidated with spectroscopic methods, using HR-MS and 1D and 2D NMR techniques, and was revealed to be oscillatoxin I, a new member of the aplysiatoxin family. Oscillatoxin I showed cytotoxicity against the L1210 mouse lymphoma cell line and diatom growth-inhibition activity against the marine diatom Nitzschia amabilis. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Review

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Review
Marine Toxins Detection by Biosensors Based on Aptamers
Toxins 2020, 12(1), 1; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12010001 - 18 Dec 2019
Cited by 9 | Viewed by 1757
Abstract
Marine toxins cause great harm to human health through seafood, therefore, it is urgent to exploit new marine toxins detection methods with the merits of high sensitivity and specificity, low detection limit, convenience, and high efficiency. Aptasensors have emerged to replace classical detection [...] Read more.
Marine toxins cause great harm to human health through seafood, therefore, it is urgent to exploit new marine toxins detection methods with the merits of high sensitivity and specificity, low detection limit, convenience, and high efficiency. Aptasensors have emerged to replace classical detection methods for marine toxins detection. The rapid development of molecular biological approaches, sequencing technology, material science, electronics and chemical science boost the preparation and application of aptasensors. Taken together, the aptamer-based biosensors would be the best candidate for detection of the marine toxins with the merits of high sensitivity and specificity, convenience, time-saving, relatively low cost, extremely low detection limit, and high throughput, which have reduced the detection limit of marine toxins from nM to fM. This article reviews the detection of marine toxins by aptamer-based biosensors, as well as the selection approach for the systematic evolution of ligands by exponential enrichment (SELEX), the aptamer sequences. Moreover, the newest aptasensors and the future prospective are also discussed, which would provide thereotical basis for the future development of marine toxins detection by aptasensors. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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Review
Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins
Toxins 2019, 11(9), 533; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins11090533 - 13 Sep 2019
Cited by 6 | Viewed by 1718
Abstract
“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable [...] Read more.
“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb. Full article
(This article belongs to the Special Issue Marine Toxins Detection)
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