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20 pages, 2472 KiB

Open AccessEditor’s ChoiceArticle

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications

by Lisbet Díaz-Asencio, Rachel J. Clausing, Mark Vandersea, Donaida Chamero-Lago, Miguel Gómez-Batista, Joan I. Hernández-Albernas, Nicolas Chomérat, Gabriel Rojas-Abrahantes, R. Wayne Litaker, Patricia Tester, Jorge Diogène, Carlos M. Alonso-Hernández and Marie-Yasmine Dechraoui Bottein

Toxins 2019, 11(12), 722; https://doi.org/10.3390/toxins11120722 - 11 Dec 2019

Cited by 34 | Viewed by 5020

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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12 pages, 1822 KiB

Open AccessEditor’s ChoiceArticle

Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay

by Jennifer R. McCall, W. Christopher Holland, Devon M. Keeler, D. Ransom Hardison and R. Wayne Litaker

Toxins 2019, 11(11), 632; https://doi.org/10.3390/toxins11110632 - 31 Oct 2019

Cited by 22 | Viewed by 4549

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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13 pages, 2171 KiB

Open AccessArticle

Chemiluminescent Receptor Binding Assay for Ciguatoxins and Brevetoxins Using Acridinium Brevetoxin-B2

by Kazuya Murata and Takeshi Yasumoto

Toxins 2019, 11(10), 580; https://doi.org/10.3390/toxins11100580 - 9 Oct 2019

Cited by 4 | Viewed by 3264

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 K_i (1.66 pM) was approximately two-fold higher than that of PbTx-3 (BTX3). In addition, the equilibrium dissociation constant (K_D) 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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11 pages, 2050 KiB

Open AccessArticle

Development of Ultra-Performance Liquid Chromatography with Post-Column Fluorescent Derivatization for the Rapid Detection of Saxitoxin Analogues and Analysis of Bivalve Monitoring Samples

by Ryuichi Watanabe, Makoto Kanamori, Hidetsugu Yoshida, Yutaka Okumura, Hajime Uchida, Ryoji Matsushima, Hiroshi Oikawa and Toshiyuki Suzuki

Toxins 2019, 11(10), 573; https://doi.org/10.3390/toxins11100573 - 1 Oct 2019

Cited by 14 | Viewed by 3931

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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12 pages, 2331 KiB

Open AccessArticle

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

by Shigeru Sato, Suzuka Takaishi, Ko Yasumoto and Shugo Watabe

Toxins 2019, 11(10), 551; https://doi.org/10.3390/toxins11100551 - 20 Sep 2019

Cited by 15 | Viewed by 3653

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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12 pages, 6543 KiB

Open AccessArticle

Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04)

by Takashi Minowa, Yuko Cho, Yasukatsu Oshima, Keiichi Konoki and Mari Yotsu-Yamashita

Toxins 2019, 11(9), 539; https://doi.org/10.3390/toxins11090539 - 16 Sep 2019

Cited by 14 | Viewed by 4802

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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10 pages, 1738 KiB

Open AccessArticle

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan

by Tsuyoshi Ikehara, Kyoko Kuniyoshi, Haruyo Yamaguchi, Yuuhiko Tanabe, Tomoharu Sano, Masahiro Yoshimoto, Naomasa Oshiro, Shihoko Nakashima and Mina Yasumoto-Hirose

Toxins 2019, 11(9), 521; https://doi.org/10.3390/toxins11090521 - 9 Sep 2019

Cited by 10 | Viewed by 3980

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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11 pages, 1494 KiB

Open AccessArticle

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels

by Keiichi Konoki, Daniel G. Baden, Todd Scheuer and William A. Catterall

Toxins 2019, 11(9), 513; https://doi.org/10.3390/toxins11090513 - 3 Sep 2019

Cited by 15 | Viewed by 4969

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 Na_v1.2, Na_v1.4 and Na_v1.5 channels showed that Na_v1.5 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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7 pages, 708 KiB

Open AccessEditor’s ChoiceCommunication

Oscillatoxin I: A New Aplysiatoxin Derivative, from a Marine Cyanobacterium

by Hiroshi Nagai, Shingo Sato, Kaori Iida, Kazutaka Hayashi, Mioko Kawaguchi, Hajime Uchida and Masayuki Satake

Toxins 2019, 11(6), 366; https://doi.org/10.3390/toxins11060366 - 21 Jun 2019

Cited by 29 | Viewed by 4177

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

Jump to: Research

22 pages, 1780 KiB

Open AccessReview

Marine Toxins Detection by Biosensors Based on Aptamers

by Wei Ye, Taomei Liu, Weimin Zhang, Muzi Zhu, Zhaoming Liu, Yali Kong and Shan Liu

Toxins 2020, 12(1), 1; https://doi.org/10.3390/toxins12010001 - 18 Dec 2019

Cited by 47 | Viewed by 6964

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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16 pages, 4513 KiB

Open AccessReview

Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

by Takeshi Tsumuraya and Masahiro Hirama

Toxins 2019, 11(9), 533; https://doi.org/10.3390/toxins11090533 - 13 Sep 2019

Cited by 18 | Viewed by 4762

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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