Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

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.

Glancing at the birth of a galaxy of scientists from Riko Majima.

Riko Majima published seven papers in this journal, and seeing these papers and their surrounding contexts allows us to glance at the birth of a galaxy of scientists.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

Cell immunolocalization of ciguatoxin-like compounds in the benthic dinoflagellate Gambierdiscus australes M. Chinain & M.A. Faust by confocal microscopy.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface.

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish.

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 µg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at -20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

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

"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.

A convergent total synthesis of the kedarcidin chromophore: 20-years in the making.

The kedarcidin chromophore is a formidible target for total synthesis. Herein, we describe a viable synthesis of this highly unstable natural product. This entailed the early introduction and gram-scale synthesis of 2-deoxysugar conjugates of both L-mycarose and L-kedarosamine. Key advances include: (1) stereoselective allenylzinc keto-addition to form an epoxyalkyne; (2) α-selective glycosylations with 2-deoxy thioglycosides (AgPF6/DTBMP) and Schmidt donors (TiCl4); (3) Mitsunobu aryl etherification to install a hindered 1,2-cis-configuration; (4) atropselective and convergent Sonogashira-Shiina cyclization sequence; (5) Ohfune-based amidation protocol for naphthoic acid; (6) Ce(III)-mediated nine-membered enediyne cyclization and ester/mesylate derivatisation; (7) SmI2-based reductive olefination and global HF-deprotection end-game. The longest linear sequence from gram-scale intermediates is 17-steps, and HRMS data of the synthetic natural product was obtained for the first time.

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons.

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins.

Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate, and quantify four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.

Synthetic Studies on Presporolide, a Putative Enediyne Precursor of Sporolides.

A synthesis of the core framework of presporolide, which possesses both a strained bicyclo[7.3.0]dodecadiyne moiety and a distinctive macrolactone structure, is reported. This synthesis features: (i) a Cu-mediated O-arylation of a hindered tertiary alcohol using triarylbismuth reagent; (ii) stereoselective construction of the strained nine-membered diyne ring; and (iii) atroposelective formation of the macrolactone.

Competent Route to Unsymmetric Dimer Architectures: Total Syntheses of (-)-Lycodine and (-)-Complanadines†A and B, and Evaluation of Their Neurite Outgrowth Activities.

Valuable synthetic routes to the Lycopodium alkaloid lycodine (1) and its unsymmetric dimers, complanadines A (4) and B (5), have been developed. Regioselective construction of the bicyclo[3.3.1]nonane core structure of lycodine was achieved by a remote functionality-controlled Diels-Alder reaction and subsequent intramolecular Mizoroki-Heck reaction. A key coupling reaction of the lycodine units, pyridine N-oxide (66) and aryl bromide (65), through C-H arylation at the C1 position of 66 provided the unsymmetric dimer structure at a late stage of the synthesis. This strategy greatly simplified the construction of the dimeric architecture and functionalization. Complanadines A (4) and B (5) were synthesized by adjusting the oxidation level of the bipyridine mono-N-oxide (67). The diverse utility of this common intermediate (67) suggests a possible biosynthetic pathway of complanadines in Nature. Both enantiomers of lycodine (1) and complanadines A (4) and B (5) were prepared in sufficient quantities for biological evaluation. The effect on neuron differentiation of PC-12 cells upon treatment with culture medium, in which human astrocytoma cells had been cultured in the presence of 1, 4, or 5 was evaluated.

Total synthesis and related studies of large, strained, and bioactive natural products.

Our chemical syntheses and related scientific investigations of natural products with complex architectures and powerful biological activities are described, focusing on the very large 3 nm-long polycyclic ethers called the ciguatoxins, highly strained and labile chromoprotein antitumor antibiotics featuring nine-membered enediyne cores, and extremely potent anthelmintic macrolides called the avermectins.

Total synthesis of avermectin B1a revisited.

Avermectins were isolated as compounds possessing anthelmintic activity from the culture broth of Streptomycesavermitilis by Omura and co-workers. Owing to their potent anthelmintic and insecticidal activities, as well as their unique pentacyclic architecture, the avermectin family attracted keen interest from synthetic organic chemists. We have recently completed a more efficient and straightforward total synthesis of avermectin B1a, as compared with previous syntheses.

Total Synthesis of Limonin.

Limonoids are highly oxygenated C13α-triterpenes and common secondary metabolites. Several hundred congeners have been isolated to date. The first total synthesis of (±)-limonin, the flagship congener of the limonoids, is now reported and features 1) a tandem radical cyclization generating the BCD ring system with the C13α configuration that is essential to the limonoids and a Robinson annulation to construct the limonoid androstane framework, 2) a singlet-oxygen cycloaddition and a Baeyer-Villiger oxidation to synthesize the highly oxidized D ring, and 3) a Suárez reaction to construct the unique AA' ring system.

Synthetic ciguatoxin CTX 3C induces a rapid imbalance in neuronal excitability.

Ciguatera is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs), sodium channel activator toxins. Symptoms of ciguatera include neurological alterations such as paraesthesiae, dysaesthesiae, depression, and heightened nociperception, among others. An important issue to understand these long-term neurological alterations is to establish the role that changes in activity produced by CTX 3C represent to neurons. Here, the effects of synthetic ciguatoxin CTX 3C on membrane potential, spontaneous spiking, and properties of synaptic transmission in cultured cortical neurons of 11-18 days in vitro (DIV) were evaluated using electrophysiological approaches. CTX 3C induced a large depolarization that decreased neuronal firing and caused a rapid inward tonic current that was primarily GABAergic. Moreover, the toxin enhanced the amplitude of miniature postsynaptic inhibitory currents (mIPSCs), whereas it decreased the amplitude of miniature postsynaptic excitatory currents (mEPSCs). The frequency of mIPSCs increased, whereas the frequency of mEPSCs remained unaltered. We describe, for the first time, that a rapid membrane depolarization caused by CTX 3C in cortical neurons activates mechanisms that tend to suppress electrical activity by shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition. Indeed, these results suggest that the acute effects of CTX on synaptic transmission could underlie some of the neurological symptoms caused by ciguatera in humans.

Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Practical route to the left wing of CTX1B and total syntheses of CTX1B and 54-deoxyCTX1B.

Ciguatoxins, the principal causative agents of ciguatera seafood poisoning, are extremely large polycyclic ethers. We report herein a reliable route for constructing the left wing of CTX1B, which possesses the acid/base/oxidant-sensitive bisallylic ether moiety, by a 6-exo radical cyclization/ring-closing metathesis strategy. This new route enabled us to achieve the second-generation total synthesis of CTX1B and the first synthesis of 54-deoxyCTX1B.

Biomimetic total synthesis of cyanosporaside aglycons from a single enediyne precursor through site-selective p-benzyne hydrochlorination.

The cyanosporasides A-F are a collection of monochlorinated benzenoid derivatives isolated from the marine actinomycetes Salinispora and Streptomyces sp. All derivatives feature one of two types of cyanocyclopenta[a]indene frameworks, which are regioisomeric in the position of a single chlorine atom. It is proposed that these chloro-substituted benzenoids are formed biosynthetically through the cycloaromatization of a bicyclic nine-membered enediyne precursor. Herein, we report the synthesis of such a bicyclic precursor, its spontaneous transannulation into a p-benzyne, and its differential 1,4 hydrochlorination reactivity under either organochlorine or chloride-salt conditions. Our bioinspired approach culminated in the first regiodivergent total synthesis of the aglycons A/F and B/C, as well as cyanosporasides D and E. In addition, empirical insights into the site selectivity of a natural-like p-benzyne, calculated to be a ground-state triplet diradical, to hydrogen, chlorine, and chloride sources are revealed.

Differential effects of ciguatoxin and maitotoxin in primary cultures of cortical neurons.

Ciguatoxins (CTXs) and maitotoxins (MTXs) are polyether ladder shaped toxins derived from the dinoflagellate Gambierdiscus toxicus. Despite the fact that MTXs are 3 times larger than CTXs, part of the structure of MTXs resembles that of CTXs. To date, the synthetic ciguatoxin, CTX 3C has been reported to activate voltage-gated sodium channels, whereas the main effect of MTX is inducing calcium influx into the cell leading to cell death. However, there is a lack of information regarding the effects of these toxins in a common cellular model. Here, in order to have an overview of the main effects of these toxins in mice cortical neurons, we examined the effects of MTX and the synthetic ciguatoxin CTX 3C on the main voltage dependent ion channels in neurons, sodium, potassium, and calcium channels as well as on membrane potential, cytosolic calcium concentration ([Ca(2+)]c), intracellular pH (pHi), and neuronal viability. Regarding voltage-gated ion channels, neither CTX 3C nor MTX affected voltage-gated calcium or potassium channels, but while CTX 3C had a large effect on voltage-gated sodium channels (VGSC) by shifting the activation and inactivation curves to more hyperpolarized potentials and decreasing peak sodium channel amplitude, MTX, at 5 nM, had no effect on VGSC activation and inactivation but decreased peak sodium current amplitude. Other major differences between both toxins were the massive calcium influx and intracellular acidification produced by MTX but not by CTX 3C. Indeed, the novel finding that MTX produces acidosis supports a pathway recently described in which MTX produces calcium influx via the sodium-hydrogen exchanger (NHX). For the first time, we found that VGSC blockers partially blocked the MTX-induced calcium influx, intracellular acidification, and protected against the short-term MTX-induced cytotoxicity. The results presented here provide the first report that shows the comparative effects of two prototypical ciguatera toxins, CTX 3C and MTX, in a neuronal model. We hypothesize that the analogies and differences in the bioactivity of these two toxins, produced by the same microorganism, may be strongly linked to their chemical structure.