RESUMEN

Neurotoxins pose significant challenges in defense and healthcare due to their disruptive effects on nervous tissues. Their extreme potency and enormous structural diversity have hindered the development of effective antidotes. Motivated by the properties of cell membrane-derived nanodiscs, such as their ultrasmall size, disc shape, and inherent cell membrane functions, here, we develop neuronal membrane-derived nanodiscs (denoted "Neuron-NDs") as a countermeasure nanomedicine for broad-spectrum neurotoxin detoxification. We fabricate Neuron-NDs using the plasma membrane of human SH-SY5Y neurons and demonstrate their effectiveness in detoxifying tetrodotoxin (TTX) and botulinum toxin (BoNT), two model toxins with distinct mechanisms of action. Cell-based assays confirm the ability of Neuron-NDs to inhibit TTX-induced ion channel blockage and BoNT-mediated inhibition of synaptic vesicle recycling. In mouse models of TTX and BoNT intoxication, treatment with Neuron-NDs effectively improves survival rates in both therapeutic and preventative settings. Importantly, high-dose administration of Neuron-NDs shows no observable acute toxicity in mice, indicating its safety profile. Overall, our study highlights the facile fabrication of Neuron-NDs and their broad-spectrum detoxification capabilities, offering promising solutions for neurotoxin-related challenges in biodefense and therapeutic applications.

Asunto(s)

Membrana Celular , Nanoestructuras , Neuronas , Neurotoxinas , Tetrodotoxina , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Animales , Ratones , Tetrodotoxina/química , Tetrodotoxina/farmacología , Neurotoxinas/química , Neurotoxinas/toxicidad , Neurotoxinas/farmacología , Nanoestructuras/química , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Toxinas Botulínicas/química , Toxinas Botulínicas/farmacología , Toxinas Botulínicas/metabolismo , Inactivación Metabólica

RESUMEN

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.

Asunto(s)

Analgésicos , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas , Tetrodotoxina , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Ratones , Tetrodotoxina/farmacología , Analgésicos/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/farmacología , Masculino , Crotalus , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Péptidos

RESUMEN

Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.

Asunto(s)

Técnicas de Placa-Clamp , Espectrometría de Masas en Tándem , Tetrodotoxina , Canales de Sodio Activados por Voltaje , Tetrodotoxina/toxicidad , Tetrodotoxina/química , Tetrodotoxina/análogos & derivados , Animales , Canales de Sodio Activados por Voltaje/metabolismo , Humanos , Ratones , Tetraodontiformes , Alimentos Marinos/análisis , Línea Celular , Cromatografía Liquida

RESUMEN

Tetrodotoxin (TTX), a lethal neurotoxin, poses a grave threat to human health. The available spectroscopic methods suffer from limitations such as complex procedures and inadequate on-site capabilities. In this study, we proposed a method using Fe3O4@Cu as a catalytic biosensor combined with SERS, colorimetry and image processing for TTX detection. Integrating the aptamer amplifies the specificity of the system and masks the catalytic activity of Fe3O4@Cu. The catalytic efficiency of Fe3O4@Cu in the H2O2-TMB reaction can quantify the concentration of TTX in the system. Consequently, oxidation of TMB (oxTMB) led to the generation and change of signals for SERS, colorimetry and image processing, enabling a three-channel quantitative detection of TTX. Under the optimal conditions, the detection limit of established SERS, colorimetry and image processing were 0.055, 2.127 and 0.243 ng/mL, respectively. This three-channel biosensor was applied to real samples, providing an accurate, stable and adaptable alternative for on-site TTX detection.

Asunto(s)

Técnicas Biosensibles , Peces , Contaminación de Alimentos , Tetrodotoxina , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Tetrodotoxina/análisis , Tetrodotoxina/química , Animales , Contaminación de Alimentos/análisis , Catálisis , Cobre/química , Cobre/análisis , Colorimetría/métodos , Espectrometría Raman/métodos , Límite de Detección , Peróxido de Hidrógeno/química , Alimentos Marinos/análisis

RESUMEN

Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.

Asunto(s)

Canales de Calcio Tipo L , Calcio , Sistema Nervioso Entérico , Motilidad Gastrointestinal , Neuronas , Tetrodotoxina , Animales , Canales de Calcio Tipo L/metabolismo , Tetrodotoxina/farmacología , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/fisiología , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Calcio/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiología , Ratones Endogámicos C57BL , Bloqueadores de los Canales de Calcio/farmacología , Femenino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Nicardipino/farmacología , Compuestos de Boro

RESUMEN

Tetrodotoxin (TTX) is a potent neurotoxin that accumulates in Takifugu rubripes, commonly known as pufferfish, through the ingestion of TTX-bearing organisms as part of their food chain. Although researchers believe that pufferfish use TTX to relieve stress, data are not currently available on how TTX affects the gut microbiota of pufferfish. To address this gap, our study aimed to investigate whether administering TTX to fish could alter their gut microbiota and overall health under various salinity conditions, including 30.0 ppt, 8.5 ppt, and 1.7 ppt salinity, which represent full-strength, isosmotic, and low-salinity stress, respectively. We analyzed the effect of TTX ingestion on the community structure, core microbiome, and metabolic capabilities of the gut microbiome using high-throughput sequencing technologies. The predominant bacterial taxa within the gut microbiome were Firmicutes (21-85%), Campilobacterota (2.8-67%), Spirochaetota (0.5-14%), and Proteobacteria (0.7-9.8%), with Mycoplasma, uncultured Arcobacteraceae, Brevinema, Vibrio, Rubritalea, and uncultured Pirellulaceae as core genera. Our findings indicated that the impact of TTX on high-abundance genera at 30.0 ppt and 8.5 ppt salinity levels was negligible, indicating their stability and resilience to TTX ingestion. However, at 1.7 ppt, TTX-fed fish showed a significant increase in uncultured Arcobacteraceae. Furthermore, our analysis of TTX-fed fish revealed taxonomic alterations in low-abundance taxa, which altered the predicted functions of the gut microbiota at all salinity levels. These results suggest that TTX administration could cause subtle effects on the metabolic functions of gut microbial communities. Overall, our study provides insights into the complex relationship between a TTX-accumulating animal, T. rubripes, and its gut microbiota.

Asunto(s)

Microbioma Gastrointestinal , Takifugu , Tetrodotoxina , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Takifugu/metabolismo , Salinidad , Bacterias/clasificación , Bacterias/genética , Bacterias/efectos de los fármacos , Bacterias/metabolismo

RESUMEN

An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE - LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9-104.2 % and 89.2-109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.

Asunto(s)

Extracción en Fase Sólida , Espectrometría de Masas en Tándem , Tetrodotoxina , Tetrodotoxina/sangre , Tetrodotoxina/orina , Extracción en Fase Sólida/instrumentación , Extracción en Fase Sólida/métodos , Cromatografía Líquida de Alta Presión , Humanos , Calibración , Sistemas en Línea , Modelos Lineales , Límite de Detección , Reproducibilidad de los Resultados

RESUMEN

Depot-type drug delivery systems are designed to deliver drugs at an effective rate over an extended period. Minimizing initial "burst" can also be important, especially with drugs causing systemic toxicity. Both goals are challenging with small hydrophilic molecules. The delivery of molecules such as the ultrapotent local anesthetic tetrodotoxin (TTX) exemplifies both challenges. Toxicity can be mitigated by conjugating TTX to polymers with ester bonds, but the slow ester hydrolysis can result in subtherapeutic TTX release. Here, we developed a prodrug strategy, based on dynamic covalent chemistry utilizing a reversible reaction between the diol TTX and phenylboronic acids. These polymeric prodrugs exhibited TTX encapsulation efficiencies exceeding 90 % and the resulting polymeric nanoparticles showed a range of TTX release rates. In vivo injection of the TTX polymeric prodrugs at the sciatic nerve reduced TTX systemic toxicity and produced nerve block lasting 9.7±2.0 h, in comparison to 1.6±0.6 h from free TTX. This approach could also be used to co-deliver the diol dexamethasone, which prolonged nerve block to 21.8±5.1 h. This work emphasized the usefulness of dynamic covalent chemistry for depot-type drug delivery systems with slow and effective drug release kinetics.

Asunto(s)

Polímeros , Profármacos , Tetrodotoxina , Profármacos/química , Profármacos/farmacología , Tetrodotoxina/química , Tetrodotoxina/toxicidad , Tetrodotoxina/administración & dosificación , Polímeros/química , Animales , Anestesia Local/métodos , Anestésicos Locales/química , Anestésicos Locales/administración & dosificación , Ácidos Borónicos/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Nervio Ciático/efectos de los fármacos , Liberación de Fármacos , Ratones

RESUMEN

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Asunto(s)

Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Oxidación-Reducción , Tetrodotoxina , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Animales , Tetrodotoxina/farmacología , Ratones , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células HEK293 , Cloraminas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Sodio/metabolismo , Potenciales de Acción/efectos de los fármacos , Compuestos de Tosilo

RESUMEN

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Asunto(s)

Cromatografía de Fase Inversa , Tetrodotoxina , Tetrodotoxina/análisis , Tetrodotoxina/química , Animales , Cromatografía Líquida de Alta Presión , Contaminación de Alimentos/análisis , Boro/química , Boro/análisis , Espectrometría de Masas en Tándem

RESUMEN

A large body of evidence has shown that treatments that interfere with memory consolidation become ineffective when animals are subjected to an intense learning experience; this effect has been observed after systemic and local administration of amnestic drugs into several brain areas, including the striatum. However, the effects of amnestic treatments on the process of extinction after intense training have not been studied. Previous research demonstrated increased spinogenesis in the dorsomedial striatum, but not in the dorsolateral striatum after intense training, indicating that the dorsomedial striatum is involved in the protective effect of intense training. To investigate this issue, male Wistar rats, previously trained with low, moderate, or high levels of foot shock, were used to study the effect of tetrodotoxin inactivation of dorsomedial striatum on memory consolidation and subsequent extinction of inhibitory avoidance. Performance of the task was evaluated during seven extinction sessions. Tetrodotoxin produced a marked deficit of memory consolidation of inhibitory avoidance trained with low and moderate intensities of foot shock, but normal consolidation occurred when a relatively high foot shock was used. The protective effect of intense training was long-lasting, as evidenced by the high resistance to extinction exhibited throughout the extinction sessions. We discuss the possibility that increased dendritic spinogenesis in dorsomedial striatum may underly this protective effect, and how this mechanism may be related to the resilient memory typical of post-traumatic stress disorder (PTSD).

Asunto(s)

Reacción de Prevención , Cuerpo Estriado , Extinción Psicológica , Ratas Wistar , Tetrodotoxina , Animales , Masculino , Extinción Psicológica/efectos de los fármacos , Extinción Psicológica/fisiología , Ratas , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Cuerpo Estriado/fisiología , Cuerpo Estriado/efectos de los fármacos , Tetrodotoxina/farmacología , Consolidación de la Memoria/efectos de los fármacos , Consolidación de la Memoria/fisiología , Amnesia/fisiopatología , Amnesia/prevención & control , Electrochoque

RESUMEN

Tetrodotoxin (TTX), a pufferfish toxin, is a highly potent neurotoxin that has been found in a wide variety of animals. The TTX-bearing flatworm Planocera multitentaculata possesses a large amount of TTX and is considered responsible for the toxification of TTX-bearing animals such as pufferfish (Takifugu and Chelonodon) and the toxic goby Yongeichthys criniger. However, the mechanism underlying TTX accumulation in flatworms remains unclear. Previous studies have been limited to identifying the distribution of TTX in multiple organs, such as the digestive organs, genital parts, and the remaining tissues of flatworms. Here, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunohistochemical staining using a monoclonal anti-TTX antibody to elucidate the detailed localization of TTX in the tissues and organs of the flatworm P. multitentaculata. Immunohistochemical staining for P. multitentaculata showed that TTX-specific signals were detected not only in the ovaries and pharynx but also in many other tissues and organs, whereas no signal was detected in the brain, Lang's vesicle, and genitalia. In addition, combined with LC-MS/MS analysis, it was revealed for the first time that TTX accumulates in high concentrations in the basement membrane and epidermis. These findings robustly support the hypotheses of "TTX utilization protection from predators."

Asunto(s)

Platelmintos , Espectrometría de Masas en Tándem , Tetrodotoxina , Animales , Tetrodotoxina/metabolismo , Tetrodotoxina/análisis , Cromatografía Liquida , Platelmintos/metabolismo , Femenino , Inmunohistoquímica , Distribución Tisular

RESUMEN

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Asunto(s)

Empalme Alternativo , Canal de Sodio Activado por Voltaje NAV1.6 , Isoformas de Proteínas , Canal de Sodio Activado por Voltaje NAV1.6/genética , Humanos , Isoformas de Proteínas/genética , Empalme Alternativo/genética , Tetrodotoxina/farmacología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Mutación , Línea Celular , Animales

RESUMEN

BACKGROUND: Tetrodotoxin (TTX) is a potent neurovirulent marine biotoxin that is present in puffer fish and certain marine animals. It is capable of causing severe neurotoxic symptoms and even death when consumed through contaminated seafood. Due to its high toxicity, developing an effective assay for TTX determination in seafood has significant benefits for food safety and human health. Currently, it remains challenging to achieve on-site determination of TTX in seafood. To facilitate mass on-site assays, more affordable technologies utilizing accessible equipment that require no skilled personnel are needed. RESULTS: A smartphone-based portable fluorescent biosensor is proposed for TTX determination by using metal-organic framework (MOF) biocomposites and cotton swabs. Oriented antibody (Ab)-decorated and fluorescent quantum dot (QD)-loaded MOF biocomposites (QD@MOF*Ab) are rapidly synthesized for binding targets and fluorescent responses by utilizing the tunability of zinc-based MOF. Moreover, facile Ab-immobilized household cotton swabs are utilized as TTX capture tools. TTX forms sandwich immune complexes with QD@MOF*Ab probes, achieving signal amplification. These probes are excited by a portable device to generate bright fluorescent signals, which can be detected by the naked eye, and TTX quantitative results are obtained using a smartphone. When observed with the naked eye, the limit of detection (LOD) is 0.4 ng/mL, while intelligent quantitation presents an LOD of 0.13 ng/mL at logarithmic concentrations of 0.2-400 ng/mL. SIGNIFICANCE: This biosensor is convenient to use, and an easy-to-operate analysis is completed within 15 min, thus demonstrating excellent performance in terms of detection speed and portability. Furthermore, it successfully determines TTX contents in puffer fish and clam samples, demonstrating its potential for monitoring seafood. Herein, this work provides a favorable rapid sensing platform that is easily portable.

Asunto(s)

Técnicas Biosensibles , Estructuras Metalorgánicas , Alimentos Marinos , Teléfono Inteligente , Tetrodotoxina , Estructuras Metalorgánicas/química , Alimentos Marinos/análisis , Técnicas Biosensibles/métodos , Tetrodotoxina/análisis , Animales , Puntos Cuánticos/química , Límite de Detección , Contaminación de Alimentos/análisis , Espectrometría de Fluorescencia , Colorantes Fluorescentes/química , Gossypium/química

RESUMEN

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

Asunto(s)

Encéfalo , Hipófisis , Saxitoxina , Takifugu , Tetrodotoxina , Animales , Tetrodotoxina/metabolismo , Hipófisis/metabolismo , Takifugu/metabolismo , Encéfalo/metabolismo , Proteínas de Peces/metabolismo , Canales de Sodio

RESUMEN

Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings further substantiate that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.

Asunto(s)

Odorantes , Neuronas Receptoras Olfatorias , Tetraodontiformes , Tetrodotoxina , Animales , Tetrodotoxina/farmacología , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/metabolismo , Odorantes/análisis , Calcio/metabolismo

RESUMEN

Fermented foods play a significant role in the human diet for their natural, highly nutritious and healthy attributes. Our aim was to study the effect of yeast extract, a fermented substance extracted from natural yeast, on colonic motility to better understand its potential therapeutic role. A yeast extract was given to rats by gavage for 3 days, and myogenic and neurogenic components of colonic motility were studied using spatiotemporal maps made from video recordings of the whole colon ex vivo. A control group received saline gavages. The yeast extract caused excitation of the musculature by increasing the propagation length and duration of long-distance contractions, the major propulsive activity of the rat colon. The yeast extract also evoked rhythmic propulsive motor complexes (RPMCs) which were antegrade in the proximal and mid-colon and retrograde in the distal colon. RPMC activity was evoked by distention-induced neural activity, but it was myogenic in nature since we showed it to be generated by bethanechol in the presence of tetrodotoxin. In conclusion, ingestion of yeast extract stimulates rat colon motility by exciting neurogenic and myogenic control mechanisms.

Asunto(s)

Colon , Motilidad Gastrointestinal , Animales , Colon/efectos de los fármacos , Colon/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Ratas , Masculino , Levaduras , Ratas Sprague-Dawley , Tetrodotoxina/farmacología

RESUMEN

For food safety, the concentrations and profiles of paralytic shellfish toxins (PSTs) and tetrodotoxin were examined in economically important scallops and bloody clams collected from the coast of the Miyagi Prefecture, Japan. PSTs were the major toxins in both species. The tetrodotoxin concentration in scallops increased in summer, although the highest value (18.7 µg/kg) was lower than the European Food Safety Authority guideline threshold (44 µg/kg). This confirmed the safety for tetrodotoxin in this area.

Asunto(s)

Bivalvos , Pectinidae , Tetrodotoxina , Animales , Tetrodotoxina/análisis , Pectinidae/química , Japón , Bivalvos/química , Toxinas Marinas/análisis , Saxitoxina/análisis , Saxitoxina/análogos & derivados , Intoxicación por Mariscos , Estaciones del Año , Contaminación de Alimentos/análisis

RESUMEN

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

Asunto(s)

Reacción en Cadena de la Polimerasa , Tetrodotoxina , Tetrodotoxina/análisis , Animales , Francia , Microscopía , Crassostrea , Código de Barras del ADN Taxonómico , Monitoreo del Ambiente/métodos , Microalgas , Estaciones del Año

RESUMEN

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Asunto(s)

Técnicas de Placa-Clamp , Tetraodontiformes , Tetrodotoxina , Tetrodotoxina/análisis , Animales , Alimentos Marinos/análisis , Ratones , Contaminación de Alimentos/análisis , Límite de Detección