Ciguatera poisoning (CP) is one of the most frequent seafood poisonings across the globe. CP results from the consumption of fish flesh that has accumulated principal toxins known as ciguatoxins (CTXs), and it mainly occurs in tropical and subtropical regions. In Japan, incidents of CP have been reported primarily from Okinawa and Amami Islands in the subtropical area. Meanwhile, there have also been reports from Mainland sporadically. Since the amount of CTXs contained in fish flesh is extremely low, a highly sensitive detection method by LC-MS/MS is required. But the currently reported detection method is applicable only to specific equipment, and many laboratories have difficulty to respond CP. In this study, to prepare for the risk of nationwide CP, we researched a universal analytical method for CTXs based on LC-MS/MS. Using a water/acetonitrile mobile phase supplemented with lithium hydroxide and formic acid gave rise to prominent peaks of the stable [M+Li]+ions. As the [M+Li]+ions did not produce valid product ions even with high collision energy, the [M+Li]+ions of each analog were set for both precursor and product ions ([M+Li]+>[M+Li]+) and monitored under the multiple reaction monitoring (MRM) mode. With the method described above, analyses of nine CTX congeners were carried out. The limit of detection (LOD, S/N>5) and quantitation (LOQ, S/N>10) were estimated as 0.005-0.030 ng/mL and 0.010-0.061 ng/mL, respectively. When the 1 mL of extract solution is prepared from 5 g of the fish tissue, the LOD and LOQ will be at 0.001-0.006 μg/kg and 0.002-0.012 μg/kg, respectively. This result indicates that we could detect the required level of 0.175 μg/kg CTX1B equivalent in fish flesh which is recommended for safe consumption in Japan. This method is considered to be a universal analytical method without depending on the specific equipment. Thus it could contribute to improving the CP investigations in nationwide laboratories.

Ciguateric syndrome is a food poisoning associated with the consumption of some species of fish that have accumulated ciguatoxins (CTXs) in their tissues. The effects of the syndrome occur with nervous imbalances which have been described for quite some time, and mentioned in sailing literature for centuries. In the last decade, research has been focused on the implementation of analytical methods for toxin identification and the study of action modes of CTXs to design effective treatments. However, an important aspect is to determine the damage that CTXs caused in the organs of affected individuals. In this work, the damages observed in tissues of mice, mainly in the small intestine, were analyzed. The animals were fed with CTX-contaminated fish muscle at concentrations 10-times below the median lethal dose (LD50) for 10 weeks. The analysis of tissues derived from the oral treatment resulted in an increased occurrence of Paneth cells, presence of lymphoid tissue infiltrating the mucosa and fibrous lesions in the mucosal layer of the small intestine. A decreasing weight in animals fed with toxic muscle was observed.

Ciguatera poisoning (CP), caused by ciguatoxins (CTXs), is one of the most common food-borne diseases, affecting more than 50,000 people each year. In most cases, CP are managed with symptomatic and supportive remedies, and no specific treatment has been devised. In this study, toward the development of therapeutic antibodies for CP, we examined to humanize mouse anti-CTX3C antibody 10C9 (m10C9), which exhibited neutralizing activity against ciguatoxin in vitro and in vivo. The complementarity determining regions were grafted onto a human germline sequence with high sequence identity to m10C9, and the backmutations were examined to maintain the binding affinity. The optimized humanized antibody, Opt.h10C9Fab, showed a strong binding affinity to CTX3C with a high affinity (KD = 19.0 nM), and only two backmutations of ArgL46 and CysH94 in the framework regions were involved in determining the antigen binding affinity.

Tropical small island developing states (SIDS), with their geographical isolation and limited resources, heavily rely on the fisheries industry for food and revenue. The presence of marine lipophilic phycotoxins (MLPs) poses risks to their economy and human health. To understand the contamination status and potential risks, the Republic of Kiribati was selected as the representative tropical SIDS and 55 species of 256 coral reef fish encompassing multiple trophic levels and feeding strategies were collected to analyze 17 typical MLPs. Our results showed that the potential risks of ciguatoxins were the highest and approximately 62% of fish species may pose risks for consumers. Biomagnification of ciguatoxins was observed in the food web with a trophic magnification factor of 2.90. Brevetoxin-3, okadaic acid, and dinophysistoxin-1 and -2 were first reported, but the risks posed by okadaic acid and dinophysistoxins were found to be negligible. The correlation analysis revealed that fish body size and trophic position are unreliable metrics to indicate the associated risks and prevent the consumption of contaminated fish. The potential risks of MLPs in Kiribati are of concern, and our findings can serve as valuable inputs for developing food safety policies and fisheries management strategies specific to tropical SIDS contexts.

Ciguatera Poisoning (CP) is an illness associated with the consumption of fish contaminated with potent natural toxins found in the marine environment, commonly known as ciguatoxins (CTXs). The risk characterization of CP has become a worldwide concern due to the widespread expansion of these natural toxins. The identification of CTXs is hindered by the lack of commercially available reference materials. This limitation impedes progress in developing analytical tools and conducting toxicological studies essential for establishing regulatory levels for control. This study focuses on characterizing the CTX profile of an amberjack responsible for a recent CP case in the Canary Islands (Spain), located on the east Atlantic coast. The exceptional sensitivity offered by Capillary Liquid Chromatography coupled with High-Resolution Mass Spectrometry (cLC-HRMS) enabled the detection, for the first time in fish contaminated in the Canary Islands, of traces of an algal ciguatoxin recently identified in G. silvae and G. caribeaus from the Caribbean Sea. This algal toxin was structurally characterized by cLC-HRMS being initially identified as C-CTX5. The total toxin concentration of CTXs was eight times higher than the guidance level proposed by the Food and Drug Administration (0.1 ng C-CTX1/g fish tissue), with C-CTX1 and 17-hydroxy-C-CTX1 as major CTXs.

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]+ > [M+Na]+). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]+ or [M+Li]+, by adding alkali metals such as Na+ or Li+ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method\'s new analytical conditions and accuracy using [M+Li]+.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced \'unknown\' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C55H79O15), 439 (C24H39O7), 149 (C10H13O), 135 (C9H11O), and 115 (C6H11O2). Additionally, characteristic product ions were observed at m/z 509 (C28H45O8), 491 (C28H43O7), 481 (C26H41O8), 463 (C26H39O7), 439 (C24H39O7), 421 (C24H37O6), 171 (C9H15O3), 153 (C9H13O2), 141 (C8H13O2), and 123 (C8H11O).

Ciguatera, a global issue, lacks adequate capacity for ciguatoxin analysis in most affected countries. The Caribbean region, known for its endemic ciguatera and being home to a majority of the global small island developing states, particularly needs established methods for ciguatoxin detection in seafood and the environment. The radioligand receptor binding assay (r-RBA) is among the in vitro bioassays currently used for ciguatoxin analysis; however, similarly to the other chemical-based or bioassays that have been developed, it faces challenges due to limited standards and interlaboratory comparisons. This work presents a single laboratory validation of an r-RBA developed in a Cuban laboratory while characterizing the performance of the liquid scintillation counter instrument as a key external parameter. The results obtained show the assay is precise, accurate and robust, confirming its potential as a routine screening method for the detection and quantification of ciguatoxins. The new method will aid in identifying high-risk ciguatoxic fish in Cuba and the Caribbean region, supporting monitoring and scientific management of ciguatera and the development of early warning systems to enhance food safety and food security, and promote fair trade fisheries.