Identification of genes associated to ß -N oxalyl- L-α, ß-diaminopropionic acid and their role in mitigating salt stress in a low-neurotoxin cultivar of Lathyrus sativus.

Grass pea has the potential to become a miracle crop if the stigma attached to it as a toxic plant is ignored. In light of the following, we conducted transcriptome analyses on the high and low ODAP-containing cultivars i.e., Nirmal and Bidhan respectively in both normal and salt stress conditions. In this study, genes that work upstream and downstream to ß-ODAP have been found. Among these genes, AAO3 and ACL5 were related to ABA and polyamine biosynthesis, showing the relevance of ABA and polyamines in boosting the ß-ODAP content in Nirmal. Elevated ß-ODAP levels in salt stress-treated Bidhan may have evolved tolerance by positively regulating the expression of genes involved in phenylpropanoid and jasmonic acid biosynthesis. Although the concentration of ß-ODAP in Bidhan increased under salt stress, it was lower than in stress-treated Nirmal. Despite this, the expression of stress-related genes that work downstream to ß-ODAP was found higher in stress-treated Bidhan. This could be because stress-treated Nirmal has lower GSH, proline, and higher H2O2, resulting in the development of severe oxidative stress. Overall, our research not only identified new genes linked with ß-ODAP, but also revealed the molecular mechanism by which a low ß-ODAP-containing cultivar developed tolerance against salinity stress.

A UPLC-MS/MS method for quantification of ß-N-methylamino-L-alanine (BMAA) in Cycas sphaerica roxb. and its use in validating efficacy of a traditional BMAA removal method.

The presence of neurotoxin ß-N-Methylamino-L-alanine (BMAA) in the seeds of Cycas sphaerica is reported for first time. We developed a UPLC-MS/MS method for BMAA quantification by derivatizing with dansyl chloride. The method successfully differentiated L-BMAA from its structural isomer 2,4-diaminobutyric acid (DAB). The extracting mixture 0.1M TCA: ACN 4:1 v/v had a recovery level of >95%. The method is a high throughput sensitive chromatographic technique with 16.42 ng g-1 Limit of Quantification. BMAA was present in the endosperm of C. sphaerica, and was not detected in the leaves and pith. Washing of seeds in running cold water for 48 h reduced BMAA content by 86%. The local communities also treat the seeds under running cold water, but only for 24 h. The results of the study thus validated the traditional BMAA removal process through cold water treatment, but recommend for increase in the treatment period to 48 h or more.

Levels of Tetrodotoxins in Spawning Pufferfish, Takifugu alboplumbeus.

Tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin thought to be used as a biological defense compound in organisms bearing it. Although TTX was thought to function as a chemical agent for defense and anti-predation and an attractant for TTX-bearing animals including pufferfish, it has recently been demonstrated that pufferfish were also attracted to 5,6,11-trideoxyTTX, a related compound, rather than TTX alone. In this study, we attempted to estimate the roles of TTXs (TTX and 5,6,11-trideoxyTTX) in the pufferfish, Takifugu alboplumbeus, through examining the location of TTXs in various tissues of spawning pufferfish from Enoshima and Kamogawa, Japan. TTXs levels in the Kamogawa population were higher than those in the Enoshima population, and there was no significant difference in the amount of TTXs between the sexes in either population. Individual differences were greater in females than in males. However, the location of both substances in tissues differed significantly between sexes: male pufferfish accumulated most of their TTX in the skin and liver and most of their 5,6,11-trideoxyTTX in the skin, whereas females accumulated most of their TTX and 5,6,11-trideoxyTTX in the ovaries and skin.

A carboxyl-functionalized covalent organic polymer for the efficient adsorption of saxitoxin.

Saxitoxin (STX), the most widely distributed neurotoxin in marine waters and emerging cyanotoxin of concern in freshwaters, causes paralytic shellfish poisoning in humans upon consumption of contaminated shellfish. To allow for the efficient monitoring of this biotoxin, it is of high importance to find high-affinity materials for its adsorption. Herein, we report the design and synthesis of a covalent organic polymer for the efficient adsorption of STX. Two ß-keto-enamine-based materials were prepared by self-assembly of 2,4,6-triformylphloroglucinol (Tp) with 2,5-diaminobenzoic acid (Pa-COOH) to give TpPa-COOH and with 2,5-diaminotoluene (Pa-CH3) to give TpPa-CH3. The carboxylic acid functionalized TpPa-COOH outperformed the methyl-bearing counterpart TpPa-CH3 by an order of magnitude despite the higher long-range order and surface area of the latter. The adsorption of STX by TpPa-COOH was fast with equilibrium reached within 1 h, and the Langmuir adsorption model gave a calculated maximum adsorption capacity, Qm, of 5.69 mg g-1, making this material the best reported adsorbent for this toxin. More importantly, the prepared TpPa-COOH also showed good reusability and high recovery rates for STX in natural freshwater, thereby highlighting the material as a good candidate for the extraction and pre-concentration of STX from aquatic environments.

Acrylamide; a neurotoxin in popcorns: a systematic review and meta-analysis.

Acrylamide is a known neurotoxic compound for humans. Foods that have high concentrations of acrylamide need to be identified. One of the food products containing acrylamide is popcorn. Popcorn is an important source of snacks for children, especially students. The presented study is a systematic review and meta-analysis of the level of acrylamide in popcorn. The search was done in different databases with the keywords; acrylamide, popcorn, popped corn. 27 articles were found by searching various databases. After initial screening and full text evaluation, 8 articles were selected for systematic review and 6 articles for meta-analysis. The amount of acrylamide in this product was in the range of 1,017.7-106 µg/kg. Microwaved corn contains lower amounts of acrylamide than other methods of preparation. The type of popcorn also had an effect on the amount of acrylamide with Meta-regression. It was found that sweet popcorn contains higher amounts of acrylamide. The overall value of acrylamide concentration in popcorns was calculated to be 459.6 ± 220.3 µg/kg. This amount is high and requires measures to reduce the amount of acrylamide.

Putative biosynthesis mechanism of the neurotoxin ß-N-methylamino-L-alanine in marine diatoms based on a transcriptomics approach.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been presumed as an environmental cause of human neurodegenerative disorders, such as Alzheimer's disease. Marine diatoms Thalassiosira minima are demonstrated here to produce BMAA-containing proteins in axenic culture while the isomer diaminobutyric acid was bacterially produced. In the co-culture with Cyanobacterium aponinum, diatom growth was inhibited but the biosynthesis of BMAA-containing proteins was stimulated up to seven times higher than that of the control group by cell-cell interactions. The stimulation effect was not caused by the cyanobacterial filtrate. Nitrogen deprivation also doubled the BMAA content of T. minima cells. Transcriptome analysis of the diatom in mixed culture revealed that pathways involved in T. minima metabolism and cellular functions were mainly influenced, including KEGG pathways valine and leucine/isoleucine degradation, endocytosis, pantothenate and CoA biosynthesis, and SNARE interactions in vesicular transport. Based on the expression changes of genes related to protein biosynthesis, it was hypothesized that ubiquitination and autophagy suppression, and limited COPII vesicles transport accuracy and efficiency were responsible for biosynthesis of BMAA-containing proteins in T. minima. This study represents a first application of transcriptomics to investigate the biological processes associated with BMAA biosynthesis in diatoms.

Assay of MAO Inhibition by Chromatographic Techniques (HPLC/HPLC-MS).

Monoamine oxidase (MAO) enzymes (MAO A and B) catalyze the oxidative deamination of biogenic amines, neurotransmitters, and xenobiotic amines and contribute to the regulation of the content of these active substances in mammalian organisms. The oxidation of biogenic amines by MAO produces hydrogen peroxide (H2O2) and aldehydes that represent risk factors for oxidative injury. The inhibitors of MAO are useful as antidepressants and neuroprotective agents. Usually, the assays of MAO determine amine deamination products or measure the H2O2 released by using direct spectrophotometric or fluorimetric methods. Direct methods are more prone to interferences and can afford inaccurate results. Those limitations can be avoided by using chromatographic techniques. This work describes a chromatographic method to assay MAO A and MAO B activity by using kynuramine as a nonselective substrate and the subsequent analysis of 4-hydroxyquinoline by RP-HPLC-DAD-fluorescence and mass spectrometry (MS). Alternatively, the assay uses the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin as a substrate of MAO that is oxidized (bioactivated) to neurotoxic pyridinium cations which are analyzed by HPLC. These methods are applied to assess the inhibition of MAO by bioactive ß-carboline alkaloids occurring in foods, plants, and biological systems.

Does Ocean Sunfish Mola spp. (Tetraodontiformes: Molidae) Represent a Risk for Tetrodotoxin Poisoning in the Portuguese Coast?

Tetrodotoxin (TTX) is a potent neurotoxin naturally occurring in terrestrial and marine organisms such as pufferfish. Due to the risk of TTX poisoning, fish of Tetraodontidae family and other puffer-related species must not be placed in the EU markets. This restriction applies to fish of the family Molidae even though no data on toxins' occurrence is available. In this study, the presence of TTX and its analogues was investigated in the main edible tissue (the white muscle) and the main xenobiotics storage organ (the liver) of ocean sunfish Mola spp. (n = 13) from the South Portuguese coast. HILIC-MS/MS analyses did not reveal TTX in the analyzed samples, suggesting an inexistent or very limited risk of TTX poisoning.

Nontoxic Enantiomeric Reference Materials for Saxitoxins.

Saxitoxin (STX) is a potent neurotoxin that is biosynthesized by toxic dinoflagellates and accumulated in shellfish via the food chain. STX and its various analogues are now monitored in shellfish by the hygiene authorities in many countries with instrumental analytical methods, which require calibration with standards. Unfortunately, STX is registered as a chemical warfare agent in Schedule 1 of the Chemical Weapons Convention, and this has made it difficult to import calibration standards into some countries. We aimed to avoid violation of the Chemical Weapons Convention and facilitate analyses by preparing calibration standards based on unnatural nontoxic antipodal STXs (ent-STXs) with the same physicochemical properties as natural STXs. Our findings demonstrate that the nontoxic ent-STXs can be safely utilized as alternative reference materials of STXs in the routine monitoring program by the local authorities and consequently can lead to reduced usage of STX.

The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom ß-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria.

ß-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype ß-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix.

Occurrence of BMAA Isomers in Bloom-Impacted Lakes and Reservoirs of Brazil, Canada, France, Mexico, and the United Kingdom.

The neurotoxic alkaloid ß-N-methyl-amino-l-alanine (BMAA) and related isomers, including N-(2-aminoethyl glycine) (AEG), ß-amino-N-methyl alanine (BAMA), and 2,4-diaminobutyric acid (DAB), have been reported previously in cyanobacterial samples. However, there are conflicting reports regarding their occurrence in surface waters. In this study, we evaluated the impact of amending lake water samples with trichloroacetic acid (0.1 M TCA) on the detection of BMAA isomers, compared with pre-existing protocols. A sensitive instrumental method was enlisted for the survey, with limits of detection in the range of 5−10 ng L−1. Higher detection rates and significantly greater levels (paired Wilcoxon's signed-rank tests, p < 0.001) of BMAA isomers were observed in TCA-amended samples (method B) compared to samples without TCA (method A). The overall range of B/A ratios was 0.67−8.25 for AEG (up to +725%) and 0.69−15.5 for DAB (up to +1450%), with absolute concentration increases in TCA-amended samples of up to +15,000 ng L−1 for AEG and +650 ng L−1 for DAB. We also documented the trends in the occurrence of BMAA isomers for a large breadth of field-collected lakes from Brazil, Canada, France, Mexico, and the United Kingdom. Data gathered during this overarching campaign (overall, n = 390 within 45 lake sampling sites) indicated frequent detections of AEG and DAB isomers, with detection rates of 30% and 43% and maximum levels of 19,000 ng L−1 and 1100 ng L−1, respectively. In contrast, BAMA was found in less than 8% of the water samples, and BMAA was not found in any sample. These results support the analyses of free-living cyanobacteria, wherein BMAA was often reported at concentrations of 2−4 orders of magnitude lower than AEG and DAB. Seasonal measurements conducted at two bloom-impacted lakes indicated limited correlations of BMAA isomers with total microcystins or chlorophyll-a, which deserves further investigation.

Local Cytotoxic Effects in Cobra Envenoming: A Pilot Study.

The cobra (genus Naja (N.)) is one of the most common venomous snakes. Due to its frequency and deadly complications of muscle paralysis, local necrosis, and chronic musculoskeletal disability, it should not be ignored. The pathology of devastating tissue destruction, even though specific antivenoms exist, is not fully clear. Here, we attempted to dig in envenomed tissues to study the clinical toxicology of cobra venom. Four cases of N. atra snake envenomation, in which the subjects developed advanced tissue injury, were involved in this study. We used enzyme-ligand sandwich immunoassay (ELISA) to assay the whole venom, cytotoxin A3 and short-chain neurotoxin (sNTX) in blood, bullae, wound discharge, and debrided tissue. We found that persistently high concentrations of venom and toxins, especially cytotoxin A3, were detected in bullae, wound discharge fluid and necrotic tissue of these patients even after large doses of specific antivenom treatment, and wide excision and advanced debridement could largely remove these toxins, lessen the size of necrosis, and promote wound healing. We also found that the point-of-care apparatus, ICT-Cobra kit, might be used to promptly monitor the wound condition and as one of the indicators of surgical intervention in cases of cobra envenomation in Taiwan.

Occurrence and Seasonal Monitoring of Domoic Acid in Three Shellfish Species from the Northern Adriatic Sea.

As filter feeders, bivalves and ascidians can accumulate contaminants present in the environment and pass them on to higher food chain levels as vectors. The consumption of bivalves contaminated with the potent neurotoxin domoic acid (DA) can cause amnesic shellfish poisoning in humans. The aim of this study was to determine seasonal differences in occurrence and accumulation of this phycotoxin in European oysters (Ostrea edulis Linnaeus, 1758) (n = 46), Queen scallops (Aequipecten opercularis Linnaeus, 1758) (n = 53), and edible ascidians of the Microcosmus spp. (n = 107), originating from the same harvesting area in the Northern Adriatic Sea. The quantification was performed using ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) preceded by derivatization with dansyl chloride. DA was found in very low concentrations throughout the year, with a maximum value of 810 µg/kg in Queen scallops. This study reveals differences in the occurrence and accumulation of DA between Queen scallops and the other two investigated species (oysters and ascidians) and the highest concentrations during the colder part of the year. Even though DA was detected in all of them, Queen scallops showed higher DA accumulation compared to the other two (p < 0.001), hence representing a sentinel species suitable for the monitoring of DA level in seafood.

The Common Sunstar Crossaster papposus-A Neurotoxic Starfish.

Saxitoxins (STXs) are a family of potent neurotoxins produced naturally by certain species of phytoplankton and cyanobacteria which are extremely toxic to mammalian nervous systems. The accumulation of STXs in bivalve molluscs can significantly impact animal and human health. Recent work conducted in the North Sea highlighted the widespread presence of various saxitoxins in a range of benthic organisms, with the common sunstar (Crossaster papposus) demonstrating high concentrations of saxitoxins. In this study, an extensive sampling program was undertaken across multiple seas surrounding the UK, with 146 starfish and 5 brittlestars of multiple species analysed for STXs. All the common sunstars analysed (n > 70) contained quantifiable levels of STXs, with the total concentrations ranging from 99 to 11,245 µg STX eq/kg. The common sunstars were statistically different in terms of toxin loading to all the other starfish species tested. Two distinct toxic profiles were observed in sunstars, a decarbomylsaxitoxin (dcSTX)-dominant profile which encompassed samples from most of the UK coast and an STX and gonyautoxin2 (GTX2) profile from the North Yorkshire coast of England. Compartmentalisation studies demonstrated that the female gonads exhibited the highest toxin concentrations of all the individual organs tested, with concentrations >40,000 µg STX eq/kg in one sample. All the sunstars, male or female, exhibited the presence of STXs in the skin, digestive glands and gonads. This study highlights that the common sunstar ubiquitously contains STXs, independent of the geographical location around the UK and often at concentrations many times higher than the current regulatory limits for STXs in molluscs; therefore, the common sunstar should be considered toxic hereafter.

Venom of the Annulated Sea Snake Hydrophis cyanocinctus: A Biochemically Simple but Genetically Complex Weapon.

Given that the venom system in sea snakes has a role in enhancing their secondary adaption to the marine environment, it follows that elucidating the diversity and function of venom toxins will help to understand the adaptive radiation of sea snakes. We performed proteomic and de novo NGS analyses to explore the diversity of venom toxins in the annulated sea snake (Hydrophis cyanocinctus) and estimated the adaptive molecular evolution of the toxin-coding unigenes and the toxicity of the major components. We found three-finger toxins (3-FTxs), phospholipase A2 (PLA2) and cysteine-rich secretory protein (CRISP) in the venom proteome and 59 toxin-coding unigenes belonging to 24 protein families in the venom-gland transcriptome; 3-FTx and PLA2 were the most abundant families. Nearly half of the toxin-coding unigenes had undergone positive selection. The short- (i.p. 0.09 µg/g) and long-chain neurotoxin (i.p. 0.14 µg/g) presented fairly high toxicity, whereas both basic and acidic PLA2s expressed low toxicity. The toxicity of H. cyanocinctus venom was largely determined by the 3-FTxs. Our data show the venom is used by H. cyanocinctus as a biochemically simple but genetically complex weapon and venom evolution in H. cyanocinctus is presumably driven by natural selection to deal with fast-moving prey and enemies in the marine environment.

Elimination of ß-N-methylamino-l-alanine (BMAA) during UV/chlorine process: Influence factors, transformation pathway and DBP formation.

As a new cyanobacterial neurotoxin generated by cyanobacteria, BMAA was closely related to amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC). In this study, the degradation of BMAA by UV/chlorine process was investigated under the impacts of chlorine dosage, NOM dosage, pH and alkalinity. Results showed that only 10% of BMAA was removed by UV irradiation and 46.8% by chlorination in 5 min, however, 98.6% of BMAA was removed by UV/chlorine process in 5 min. The reaction rates were increased under alkaline conditions, but all achieved complete degradation in 5 min. Besides, HCO3- had slight inhibition, while NOM had significant inhibition on the degradation of BMAA. Furthermore, based on the detected degradation products of BMAA during UV/chlorine process, the possible degradation pathways were concluded. Overall, outcomes of this study exhibited that the use of the UV/chlorine process for BMAA degradation was appropriate in practical applications.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Awareness on tetrodotoxin of illegal activity: forensic issue from a rare homicide case report and literature review.

The authors describe an extremely rare case of homicide by injecting tetrodotoxin (TTX) as lethal neurotoxin found in puffer fish. After a thorough investigation, the male victim was found to have a broken stalk from syringe needle in the subcutaneous tissue of left buttock and severe asphyxia confirmed by the main pathological findings at autopsy. During tortuous toxicological analysis，TTX was revealed by ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) as well as acute intoxication confirmed from forensic examination. The literature of qualitative and quantitative determination of TTX from human fluids was also reviewed to expect widely acceptable detection strategies. This case highlighted the importance of TTX toxicant with chemical formula name purchased through e-commerce，so as to improve particular emphasis and supervision on harmful substances possibly using hidden information or illegal means. Histopathological and toxicological results demonstrated here provided a reference and other useful information to the challenges of forensic casework. In general, the case report illustrates medico-legal issues of more attention to the possibility of TTX poisoning in rapid death and the need of routine postmortem tox screening in future practice.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded ß-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Toxicity and bioaccumulation of two non-protein amino acids synthesised by cyanobacteria, ß-N-Methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), on a crop plant.

In order to study the toxicity of the cyanobacterial non-protein amino acids (NPAAs) L-ß-N-methylamino-L-alanine (BMAA) and its structural isomer L-2,4-diaminobutyric acid (DAB) in the forage crop plant alfalfa (Medicago sativa), seedlings were exposed to NPAA-containing media for four days. Root growth was significantly inhibited by both treatments. The content of derivatised free and protein-bound BMAA and DAB in seedlings was then analysed by LC-MS/MS. Both NPAAs were detected in free and protein-bound fractions with higher levels detected in free fractions. Compared to shoots, there was approximately tenfold more BMAA and DAB in alfalfa roots. These results suggest that NPAAs might be taken up into crop plants from contaminated irrigation water and enter the food chain. This may present an exposure pathway for NPAAs in humans.