On the structure and stability of novel cationic DPPC liposomes doped with gemini surfactants

A novel formulation of cationic liposomes was studied by mixing dipalmitoylphosphatidylcholine (DPPC) with tetradecyltrimethylammonium bromide gemini surfactants with different alkane spacer groups lengths attached to their ammonium head-groups. The physicochemical characterization of the cationic liposomes was obtained by combining experimental results from differential scanning microcalorimetry (DSC) with molecular dynamic simulations, in order to understand their structural configuration. An adapted Ising model was used to interpret the results in terms of cooperativity of the phase transitions. The gemini surfactants partition into the lipid bilayer of DPPC liposomes, and the induced changes in colloidal stability and phase transition were analyzed in detail. The DPPC liposomes became positively charged upon gemini surfactant partition, showing increased colloidal stability. Our results show significant differences in structural configuration between gemini surfactants with short and long spacer lengths. While gemini with shorter spacers allocate within the lipid bilayer with both headgroups in the same layer, geminis with longer spacers unexpectedly intercalate in the lipid membrane in a particular zig-zag configuration, with each headgroup located at a different side of the bilayer, altering the coupling degree parameters of the membrane’s phase transition. The extraordinary increase of colloidal stability of DPPC liposomes with gemini surfactants at very low molar ratio and the possibility to tune the physicochemical properties of the membrane by control de spacer length of the geminis opens new possibilities for cationic liposomal formulations with potential applications in vaccines, drug/gene delivery or biosensingThis work was supported by the Spanish Research Agency (AEI) under Project PID2019-109517RB-I00. ERDF funds are also acknowledged. Facilities provided by the Galician Supercomputing Centre (CESGA) are also acknowledgedS

In silico simulations and functional cell studies evidence similar potency and distinct binding of pacific and caribbean ciguatoxins

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scaleOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER-co-funded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01, ED431C 2021/29, and the Centro singular de investigación de Galicia accreditation 2019–2022 ED431G 2019/03). From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX, and the EUROCIGUA project: “Risk Characterization of Ciguatera Fish Poisoning in Europe” GP/EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA). From Ministerio de Ciencia e Innovación PID2020-115010RB-I00/AEI/10.13039/501100011033. David Castro (D.C.) financial support for the PhD studies was obtained through EUROCIGUA project: Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/AFSCO/2015/03, co-funded by the EFSA. Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207S

Desenvolvemento e optimización de LC-MS para a identificación e confirmación de ciguatoxinas

CFP (Ciguatera Fish Poissoning) is a disease associated with the appearance of gastrointestinal and neurological symptoms caused by the consumption of fish contaminated with ciguatoxin (CTX), whose precursors are produced by benthic dinoflagellates of the genus Gambierdiscus. Although these CTXs producing species are endemic to coral reefs in the Pacific, Indian and Caribbean marine regions, cases of CFP have recently been confirmed in the Canary Islands and other European regions, in addition to the identification of various species of Gambierdiscus in the North Atlantic, representing an emerging problem for public health in Europe. The lipophilicity of the mentioned toxins coupled with the complexity of the matrix in which they are present necessitates the optimization of efficient extraction and purification steps for the removal of endogenous matrix compounds which would affect the efficiency of the analysis. The PhD thesis will focus on the design and evaluation of effective sample preparation protocols for the analysis of ciguatoxins in contaminated fish.La CFP (Ciguatera Fish Poissoning) es una enfermedad asociada a la aparición de síntomas gastrointestinales y neurológicos provocados por el consumo de peces contaminados con ciguatoxina (CTX), cuyos precursores son producidos por dinoflagelados bentónicos del género Gambierdiscus. Aunque estas especies productoras de CTXs son endémicas de los arrecifes de coral de las regiones marinas del Pacífico, Índico y Caribe, recientemente se ha confirmado casos de CFP en las Islas Canarias y otras regiones europeas, además de haberse identificado diversas especies de Gambierdiscus en el Atlántico Norte, representando un problema emergente para la salud pública en Europa. El carácter lipofílico de las citadas toxinas unido a la complejidad de la matriz en las que se encuentran presentes hace necesario la optimización de etapas de extracción y purificación eficaces para la eliminación de los compuestos endógenos de la matriz que afectarían a la eficacia del análisis. La Tesis doctoral se centrará en el diseño y evaluación de protocolos de preparación de muestra eficaces para el análisis de ciguatoxinas en peces contaminados.A CFP (Ciguatera Fish Poissoning) está asociado coa aparición de síntomas gastrointestinais e neurolóxicos causados polo consumo de peixes contaminados con ciguatoxinas (CTX), cuxos precursores son producidos por dinoflaxelados bentónicos do xénero Gambierdiscus. Aínda que estas especies produtoras de CTXS son endémicos dos arrecifes de corais das rexións mariñas do Pacífico, Índico e Caribe, recentemente confirmaronse casos de CFP nas Illas Canarias e outras rexións europeas, así como varias especies de Gambierdiscus foron identificados no Atlántico Norte, o que representa un problema emerxente para a saúde pública en Europa. O carácter lipofílico destas toxinas combinadas coa complexidade da matriz en que están presentes require unha optimización de etapas de extracción e purificación eficaces para a eliminación de compostos endóxenos da matriz que afectan a eficacia da análise. A tese de doutoramento terá como foco o deseño e avaliación de protocolos de preparación de mostra eficaces para a análise da ciguatoxinas en peixes contaminados

Liquid chromatography coupled to high-resolution mass spectrometry for the confirmation of Caribbean Ciguatoxin-1 as the main toxin responsible for Ciguatera poisoning caused by fish from European Atlantic coasts

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species (Seriola fasciata) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands (Portugal).European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03Xunta de Galicia | Ref. ED481A-2018/20

Preparation of ciguatoxin reference materials from Canary Islands (Spain) and Madeira Archipelago (Portugal) fish

Ciguatoxins (CTXs) are naturally occurring neurotoxins that can accumulate in fish and cause Ciguatera Poisoning (CP) in seafood consumers. Ciguatoxic fish have been detected in tropical and subtropical regions of the world including the Pacific and Indian Oceans, the Caribbean Sea, and more recently in the northeast Atlantic Ocean. The biogeographic distribution of ciguatoxic fish appears to be expanding; however, the paucity of CTX standards and reference materials limits the ability of public health authorities to monitor for these toxins in seafood supply chains. Recent reports establish that Caribbean Ciguatoxin-1 (C-CTX1) is the principal toxin responsible for CP cases and outbreaks in the northeast Atlantic Ocean and that C-CTX congener profiles in contaminated fish samples match those from the Caribbean Sea. Therefore, in this work, C-CTX reference materials were prepared from fish obtained from the northeast Atlantic Ocean. The collection of fish specimens (e.g., amberjack, grouper, or snapper) was screened for CTX-like toxicity using the in vitro sodium channel mouse neuroblastoma cytotoxicity assay (N2a cell assay). Muscle and liver tissues from toxic specimens were pooled for extraction and purified products were ultimately profiled and quantified by comparison with authentic C-CTX1 using LC-MS/MS. This work presents a detailed protocol for the preparation of purified C-CTX reference materials to enable continued research and monitoring of the ciguatera public health hazard. To carry out this work, C-CTX1 was isolated and purified from fish muscle and liver tissues obtained from the Canary Islands (Spain) and Madeira archipelago (Portugal).European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03Xunta de Galicia | Ref. ED481A-2018/20

Accumulation of C-CTX1 in muscle tissue of goldfish (Carassius auratus) by dietary experience

Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g−1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g−1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.Ministerio de Ciencia e Innovación | Ref. PID2019 108781RR C22European Food Safety Authority | Ref. GP/EFSA/AFSCO/201

Ciguatoxin detection in flesh and liver of relevant fish species from the Canary Islands

The Canary Islands are a ciguatoxin (CTX) hotspot with an established official monitoring for the detection of CTX in fish flesh from the authorised points of first sale. Fish caught by recreational fishermen are not officially tested and the consumption of toxic viscera or flesh could lead to ciguatera poisoning (CP). The objectives of this study were to determine the presence of CTX-like toxicity in relevant species from this archipelago, compare CTX levels in liver and flesh and examine possible factors involved in their toxicity. Sixty amberjack (Seriola spp.), 27 dusky grouper (Epinephelus marginatus), 11 black moray eels (Muraena helena) and 11 common two-banded seabream (Diplodus vulgaris) were analysed by cell-based assay (CBA) and Caribbean ciguatoxin-1 (C-CTX1) was detected by liquid chromatography mass spectrometry (LC-MS/MS) in all these species. Most of the liver displayed higher CTX levels than flesh and even individuals without detectable CTX in flesh exhibited hepatic toxicity. Black moray eels stand out for the large difference between CTX concentration in both tissues. None of the specimens with non-toxic liver showed toxicity in flesh. This is the first evidence of the presence of C-CTX1 in the common two-banded seabream and the first report of toxicity comparison between liver and muscle from relevant fish species captured in the Canary Islands.European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03Ministerio de Educación | Ref. FPU17/04022Ministerio de Ciencia e Innovación | Ref. PID2019-108781RR-C21Ministerio de Ciencia e Innovación | Ref. PID2019 108781RR C22Xunta de Galicia | Ref. ED481A-2018/20

Use of mass spectrometry to determine the diversity of toxins produced by Gambierdiscus and Fukuyoa species from Balearic Islands and Crete (Mediterranean Sea) and the Canary Islands (Northeast Atlantic)

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions.European Food Safety Authority | Ref. EuroCigua (GP/EFSA/AFSCO/2015/03)Xunta de Galicia | Ref. ED481A-2018/207IRTA-URV-Santander | Ref. 2016 PMF-PIPF-7

Toxicity screening of a Gambierdiscus australes strain from the Western Mediterranean sea and identification of a novel maitotoxin analogue

Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions.European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03Xunta de Galicia | Ref. ED481A-2018/207Generalitat de Catalunya | Ref. CERC