Tetrodotoxins (TTXs) are a group of potent neurotoxins produced by bacteria, usually found in pufferfish. These toxins may reach consumers and cause them an intoxication with symptoms such as muscle weakness, respiratory failure, and ultimately death. Therefore, the detection of TTXs is of great importance in seafood safety. The cell-based assay (CBA) is commonly used to detect TTXs because of its high sensitivity and because it provides an estimation of the composite toxicity of a sample. However, when analysing natural seafood samples, matrix effects may interfere in the CBA. In this work, five insoluble cyclodextrin polymers (βCDPi-1.4SO₃ (DS2), βCDPi-1.3SO₃ (DS2), βCDPi-1.3SO₃ (DS4), βCDPi-1.4SO₃ (DS4) and βCDPi-NCH₃ (DS5)) have been investigated as novel clean-up materials for natural extracts containing TTXs. The clean-up method involved an incubation of the cyclodextrin polymers with TTX standard (at different concentrations), followed by an extraction of the TTXs captured by the cyclodextrins allowing it to bind with TTX. The purified TTXs were then detected using a CBA, since these toxins act on voltage-gated sodium channels. The best recoveries were achieved with βCDPi-1.4SO₃ (DS2) (~80%). This cyclodextrin polymer had the capability of capturing as much as ~20 ng TTX/mg cyclodextrin polymer. Therefore, the developed clean-up method combined with CBA is a promising strategy for the detection of low TTXs contents in pufferfish and shellfish extracts, which could be implemented in research and monitoring programs for seafood safety.

Ciguatoxins (CTXs) are lipophilic marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. The ingestion of fish contaminated with CTXs causes ciguatera fish poisoning (CFP), the most relevant seafood-borne disease worldwide. CFP is characterized by severe neurological, gastrointestinal and cardiovascular disorders, and affects approximately between 50,000 and 500,000 consumers annually worldwide. The cell-based assay (CBA) is commonly used for the detection of CTXs because it is highly sensitive and provides toxicological information. However, fish matrix compounds may interfere in the CBA. In this work, three different monoclonal antibodies (mAbs), two capture (3G8 and 10C9) and a detector (8H4) conjugated to magnetic beads have been evaluated as clean-up materials and, at the same time, as tools to pre-concentrate CTXs present in fish extracts prior to analysis with CBA. The applicability of the system was firstly demonstrated with fish extracts spiked with CTX congeners (CTX1B and CTX3C) and, afterwards, with extracts from naturally contaminated fish containing CTXs. The analysis of samples showed that the antibody-magnetic bead conjugates provide good toxin recoveries and allow exposing neuro-2a cells to higher fish tissue concentrations. This clean-up and pre-concentration strategy decreases the limit of detection of the CBA, which is a great achievement considering the extremely low CTX contents that need to be detected to guarantee fish safety and protect human health.

INTRODUCTION

Snakebite envenoming is of high priority issue in many tropical and subtropical regions of the world; more than 150000 are killed each year from snakebites while many millions survive from envenoming but face many disabilities and long-term health consequences. Antivenom serums are the only treatment for snakebite envenoming however they present a number of major associated problems, several adverse reactions and their availability is limited. In addition, administration is frequently based on symptomatology resulting in wasting crucial time.

In this study, we investigate the cytotoxicity of Macrovipera Lebetina Lebetina (Cypriot endemic viper) venom and examine the antivenom potency of natural compounds (peptides and endemic plant extracts).

RESULTS

Two different cell lines are used for testing the venom toxicity, human keratinocytes (HaCaT) and human cancer cell line (A549). Venom exhibits great cytotoxicity (24 h) against both cell lines: in HaCaT cells levels reach up to approximately 30% (10 mg/ml) while in A549 cells venom kills almost the 95% cells (5 mg/ml), respectively.

Peptides and plant extracts are tested for their antivenom potency by treating cells at 3 and 6 hours after adding venom in various concentrations. Before testing for their antivenom potency the compounds have been examined for their cytotoxicity with MMT assay in four different concentrations.

Reversed effect is significantly present underlining the cytoprotective effect of natural peptides and plant extracts against the venom of Macrovipera Lebetina Lebetina.

Acknowledgements: We thank Cypriot Start-Up MedVenom Ltd, for providing the venom of Macrovipera Lebetina Lebetina

Ciguatoxins (CTXs) are potent marine neurotoxins that cause ciguatera fish poisoning (CFP). CFP occurs mainly in the tropical and subtropical Indo-Pacific region and Caribbean Sea. However, their presence, toxicity and toxin profile have been poorly described in the Indian Ocean.

In this study, the liver, muscle, and viscera of four fish specimens of three different species (one Lutjanus bohar, two Variola louti and one Carcharinus leucas) caught in the waters of La Réunion Island were analysed. One of the V. louti was obtained after a CFP incident. Toxicity was analysed by screening CTX-like toxicity with the neuro-2acell-based assay. Their toxicity values ranged between 0,013 to 1,26 µg equiv CTX1B/kg in flesh, 2,55 to 16,40 equiv CTX1B/kg in liver and 0,51 to 3,12 equiv CTX1B/kg in viscera. In general, liver and viscera showed higher concentrations of CTX-like compounds than flesh.

The three fish tissue extract clean-up were evaluated using the combination of Florisil and C18 SPE cartridges, recoveries of CTX1B were between 48,6 and 89.7% depending on the tissue. Additionally, a new screening method using a UPLC coupled with a Xevo TQXS for CTXs was optimized at 11 minutes per sample and in which a total of fourteen analogues, with three transitions each one, were included. This method together with three other methods including more than 30 known CTX congeners into the LC-MS/MS were used.

The toxin profile for the four fish specimens were evaluated using liquid chromatography coupled to mass spectrometry (LC-MS/MS) and to high resolution mass spectrometry (LC-HRMS). Several potential CTX congeners were identified: the flesh of the V.louti after a CFP incident confirmed the presence of CTX1B, (0.12 µg equiv CTX1B/kg), 52-epi-54-doxyCTX1B and 54-deoxyCTX1B.. In the second V. louti individual and the L. bohar, CTX1B was identified in the three tissues. The C. leucas samples revealed the presence of a complex CTX contaminant profile showing congeners of CTX3C-group.

Endemic species represent a great untapped resource for a variety of bioactive agents that may have great potential for many applications. In addition, a better understanding of envenomation's pathogenic mechanisms would allow the exploration of approaches to deal with this event. It has been reported that venoms are a pool of antimicrobial peptides, including against clinically relevant Gram positive and Gram negative bacteria. Many studies have also demonstrated the effect of venoms on hemolysis, inflammatory responses and the activation of the complement system. We sought to study the effects of venoms from Cypriot endemic species, the Cypriot blunt-nosed viper, Macrovibera l. lebetina and the scorpion Mesobuthus cyprius. It was found that both crude venoms at different concentrations, promote significant degradation of the C3 complement component, as shown by SDS-PAGE analysis, also generating anaphylatoxins. Furthermore, the venoms were tested for their antimicrobial activity against Staphylococcus epidermidis, Streptococcus pneumoniae and Escherichia coli via the microdilution method at a final concentration range of 10-300 μg/ml. Both crude venoms did not display any antimicrobial activity against the tested organisms. Further characterization and investigation of selected peptides and proteins is underway for their antimicrobial activity and effects on other complement components.

Russell’s viper (Daboia siamensis) is a medically important snake in Myanmar due to its high morbidity and mortality. The genome of Myanmar Russell’s viper has not sequenced until recently, thus RNA-Sequencing has been used to predict genes encoding this snake’s toxins. Venom glands were dissected from four adult D. siamensis specimens (two males and two females) provided by a local Myanmar Snake Farm. mRNA was extracted and sequenced on the Illumina HiSeq platform, then assembled de novo using the Trinity software. Candidate toxin genes were identified using the Venomix pipeline and their expression levels were calculated by mean of RSEM software. Identified toxin candidates were aligned with previously described venom proteins using Clustal Omega. Candidate venom transcripts were classified into 23 toxin gene families, which included 53 unique transcripts identified as full-length sequences. Among them, 39 full-length sequences represented the nine newly identified toxin gene families in D. siamensis: Neprilysin (2), Cystatin (5), Latrotoxins (11), Waprin (1), Vipericidin (1), Veficolin (1), Endothelial lipases (9), Vespryn (ohanin) (8) and three finger toxins (1). Their expression levels were found to be moderate to low (TPM= 1.49 to 213.37). The majority of the toxin candidates were resembled to typical toxins found in elapids, with a majority of the toxins exhibiting neurotoxic activity and tissue damage. A smaller proportion of candidate toxin transcripts were predicted to exhibit antimicrobial activity and anti-metastatic effect. Our results indicate their functional activities could be studied further for therapeutic applications.

From its lethal sting to its medicinal potential, scorpion venom is a paradoxical elixir of nature. This venom contains a variety of molecules, including enzymes, molecules involved in allergic reactions, and, most notably, neurotoxins that exert a potent pharmacological activity on the membrane of ion channels of excitable cells. In this study, we conducted an exhaustive analysis of the venoms derived from Moroccan scorpions belonging to the Androctonus genera. Using electrospray mass spectrometry coupled with high-performance liquid chromatography (HPLC), we aimed to provide detailed information regarding the composition and molecular nature of these venoms. We initially fractionated the venoms using HPLC, and the fractions were subsequently analyzed via mass spectrometry to establish peptide maps and fingerprints for each venom. We obtained a wide range of molecular weights, varying between 236 and 622, depending on factors such as species, genus, geographical location, age, sex, and diet. Our analysis revealed that the molecular masses of the venoms were predominantly found between 2000 and 4000 Da and corresponded mainly to short toxins that blocked the K⁺ channels. Larger molecular weights exceeding 4000 Da were identified as long toxins modulating the Na⁺ channels. Moreover, we discovered other peptides below 2000 Da that were previously unknown. Our observations also indicated that the venom of the Androctonus genera contained higher quantities of masses corresponding to both long and short toxins. This study provides valuable insight into the complex composition of scorpion venoms and underscores the significance of understanding their composition in the development of novel therapeutic approaches.

Venomous snake species can be broadly grouped into three families: Colubridae, Elapidae, and Viperidae. In Portugal, there are four venomous species: the horned viper (Vipera latastei), seoane (Vipera seoanei), rat snake (Malpolon monspessulanus) and the hooded snake (Macroprotodon brevis ibericus), and in the UK the common European adder (Vipera berus). They inoculate the venom through specialised fangs located anteriorly in the maxilla, associated with a venom gland, at high pressure by compression of the gland by skeletal muscle fibres. Snake venom is a complex mixture of toxins whose composition varies depending on the families, genera, species and even subspecies. In Europe, particularly Portugal, there are no published data on the frequency of these types of incidents, but it is estimated to be a high incidence, mostly in dogs. Thus, to characterise the injuries caused by the bite of venom snacks in domestic animals, the authors describe cases in dogs, cats and goats with a suspected snack bite. Animals present wounds compatible with snakebites, and two points 1 to 1.8 cm apart that could be noted in the head or limbs. The main clinical signs observed included pain, oedema and necrosis. From the animals that died the post-mortem examination revealed subcutaneous and muscular necrosis and haemorrhages of surrounding tissues, including muscles and organic haemorrhage.
The severity of envenomation will depend on the quantity of inoculated venom, the species, age, size and previous state of health of the bitten animal, bite location and post-bite excitability. Small animals and young animals may be more susceptible to the effects of venom than larger, healthier animals. Livestock animals may be particularly vulnerable to the effects of venom, as they are often exposed to snakes while grazing in fields or pastures.
With this study, the authors hope to help improve the knowledge regarding snakebites in Europe.

Pteridium aquilinum (L.) Kuhn, commonly known as bracken-fern, is a cosmopolitan plant species absent only from polar and desert regions. Worldwide, Pteridium aquilinum has been rapidly spreading due to the neglect of agricultural fields and the growing wave of forest fires. This plant has several toxic components such as thiaminases, brains, active glucosides, beta-glucopyranosides and ptaquloside in its constitution that can be lethal to animals. The animals do not normally choose it as food, except in situations of food shortage, in periods of drought, when the ferns are still green or when the animals graze in places with large amounts of the plant in question. In order to revisit the major syndromes caused be P. aquilinum major toxins, the authors present several cases of acute and chronic ruminant poisoning associated with ingesting P. aquilinum in Portugal in sheep and cattle. The primarily associated syndromes observed in animals that consume this plant were thiamine deficiency, blindness in sheep, acute or subacute poisoning with bone marrow depression, and consequently, leucopenia and bladder tumours in cattle. Given the economic impact of the diseases caused by the toxins of this plant, and possible public health implications, it is imperative to minimise the syndromes in animals and establish measures for their prevention.

The growing interest in herbal and alternative medicines demands information on the toxicity risk assessment of the various plant extracts used in traditional medicines. The presence of a potential toxic phytochemicals in Tithonia diversifolia extracts necessitates toxicological evaluation of its biologically active constituents. The study evaluated the in vivo sub-chronic toxicity of the moderately polar fractions of T. diversifolia in a rat model. The ethyl acetate soluble portion from the methanol extract was separated by vacuum liquid chromatographic method. Three-dose levels- an observed adverse effect level (OAEL) of 2000 mg/kg, a no-observed adverse effect level (NOAEL) of 80 mg/kg and an intermediate dose of 500 mg per kg body weight of rats per day- were selected for a 28-day repeated dosing for the sub-chronic toxicological evaluation. The LC-MS dereplication of the active fractions showed the presence of sesquiterpene lactones such as diversifolin, diversifolin methylether, tagitinin A, tagitinin C-F, woodhousin, and orizatin and many unidentified peaks. There was a significant reduction (p < 0.05) in the weights of and food consumption by the rats dosed with OAEL of the fraction on week 1 which normalized during the subsequent weeks of the study. The histopathological examination showed a mild necrosis and degeneration of hepatocytes in the centrilobular areas of the rats treated with OAEL of the active VLC fraction. There was no T. diversifolia-related adverse toxicological events in rats with a 2000 mg/kg/day when dosed orally for 28 days.