An exploration of the binding prediction of anatoxin-a and atropine to acetylcholinesterase enzyme using multi-level computer simulations.

Acetylcholinesterase (AChE) is crucial for the breakdown of acetylcholine to acetate and choline, while the inhibition of AChE by anatoxin-a (ATX-a) results in severe health complications. This study explores the structural characteristics of ATX-a and its interactions with AChE, comparing to the reference molecule atropine for binding mechanisms. Molecular docking simulations reveal strong binding affinity of both ATX-a and atropine to AChE, interacting effectively with specific amino acids in the binding site as potential inhibitors. Quantitative assessment using the MM-PBSA method demonstrates a significantly negative binding free energy of -81.659 kJ mol-1for ATX-a, indicating robust binding, while atropine exhibits a stronger binding affinity with a free energy of -127.565 kJ mol-1. Umbrella sampling calculates the ΔGbindvalues to evaluate binding free energies, showing a favorable ΔGbindof -36.432 kJ mol-1for ATX-a and a slightly lower value of -30.12 kJ mol-1for atropine. This study reveals the dual functionality of ATX-a, acting as both a nicotinic acetylcholine receptor agonist and an AChE inhibitor. Remarkably, stable complexes form between ATX-a and atropine with AChE at its active site, exhibiting remarkable binding free energies. These findings provide valuable insights into the potential use of ATX-a and atropine as promising candidates for modulating AChE activity.

Liquid chromatography-high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products.

Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC-HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,ß-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,ß-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously.

Removal of saxitoxin and anatoxin-a by PAC in the presence and absence of microcystin-LR and/or cyanobacterial cells.

Cyanobacteria can produce cyanotoxins such as microcystin-LR (MC), saxitoxin (STX), and anatoxin-a (ANTX-a) which are harmful to humans and other animals. Individual removal efficiencies of STX and ANTX-a by powdered activated carbon (PAC) was investigated, as well as when MC-LR and cyanobacteria were present. Experiments were conducted with distilled water and then source water, using the PAC dosages, rapid mix/flocculation mixing intensities and contact times of two drinking water treatment plants in northeast Ohio. At pH 8 and 9, STX removal was 47%-81% in distilled water and 46%-79% in source water, whereas it was 0-28% for pH 6 in distilled water and 31%-52% in source water. When 1.6 µg/L or 20 µg/L MC-LR was present with STX, STX removal was increased with PAC simultaneously removing 45%-65% of the 1.6 µg/L MC-LR and 25%-95% of the 20 µg/L MC-LR depending on the pH. ANTX-a removal at pH 6 was 29%-37% for distilled water and 80% for source water, whereas it was 10%-26% for pH 8 in distilled water and 28% for pH 9 in source water. The presence of cyanobacteria cells decreased ANTX-a removal by at least 18%. When 20 µg/L MC-LR was present with ANTX-a in source water, 59%-73% ANTX-a and 48%-77% of MC-LR was removed at pH 9 depending on the PAC dose. In general, a higher PAC dose led to higher cyanotoxin removals. This study also documented that multiple cyanotoxins can be effectively removed by PAC for water at pH's between 6 and 9.

Anatoxin-a: Overview on a harmful cyanobacterial neurotoxin from the environmental scale to the molecular target.

Anatoxin-a (ATX-a) is a neurotoxic alkaloid, produced by several freshwater planktonic and benthic cyanobacteria (CB). Such CB have posed human and animal health issues for several years, as this toxin is able to cause neurologic symptoms in humans following food poisoning and death in wild and domestic animals. Different episodes of animal intoxication have incriminated ATX-a worldwide, as confirmed by the presence of ATX-a-producing CB in the consumed water or biofilm, or the observation of neurotoxic symptoms, which match experimental toxicity in vivo. Regarding toxicity parameters, toxicokinetics knowledge is currently incomplete and needs to be improved. The toxin can passively cross biological membranes and act rapidly on nicotinic receptors, its main molecular target. In vivo and in vitro acute effects of ATX-a have been studied and make possible to draw its mode of action, highlighting its deleterious effects on the nervous systems and its effectors, namely muscles, heart and vessels, and the respiratory apparatus. However, very little is known about its putative chronic toxicity. This review updates available data on ATX-a, from the ecodynamic of the toxin to its physiological and molecular targets.

First Evidence of the Presence of Anatoxin-A in Sea Figs Associated with Human Food Poisonings in France.

From January 2011 to March 2018, 26 patients aged from 20 to 80 years old reported being sick in France after eating sea figs of the genus Microcosmus. The patients had symptoms evoking a cerebellar syndrome: blurred or double vision, ataxia and dizziness, asthenia, headache, muscle cramps, paresthesia and digestive disorders (nausea, vomiting and diarrhea). Three of the 18 food poisoning events recorded by the Poison Control Center in Marseille and involving four patients were further investigated as the meal leftovers were collected and analyzed. A previous study ruled out the presence of the regulated lipophilic marine toxins after high-resolution mass spectrometry, but further analyses were required to look for hydrophilic cyanotoxins. The sea fig leftovers from food poisoning case Numbers 1 (January 2011), 6 (December 2012) and 17 (March 2018) of this published case series were analyzed by hydrophilic interaction liquid chromatography coupled to low- and high-resolution mass spectrometry to investigate the presence of hydrophilic cyanotoxins. The sea fig samples showed anatoxin-a (ATX-a) concentrations ranging from 193.7 to 1240.2 µg/kg. The sea fig control sample analyzed was also contaminated with ATX-a but in a much smaller concentration (22.5 µg/kg). To the best of our knowledge, this is the first report of human food poisoning involving ATX-a as the possible causative toxin where the cyanotoxin could be unequivocally identified.

Neurotoxic effects of sublethal concentrations of cyanobacterial extract containing anatoxin-a(s) on Nauphoeta cinerea cockroaches.

The detection of cyanotoxins, such as the anatoxin-a(s), is essential to ensure the biological safety of water environments. Here, we propose the use of Nauphoeta cinerea cockroaches as an alternative biological model for the biomonitoring of the activity of anatoxin-a(s) in aquatic systems. In order to validate our proposed model, we compared the effects of a cyanobacterial extract containing anatoxin-a(s) (CECA) with those of the organophosphate trichlorfon (Tn) on biochemical and physiological parameters of the nervous system of Nauphoeta cinerea cockroaches. In brain homogenates from cockroaches, CECA (5 and 50 µg/g) inhibited acetylcholinesterase (AChE) activity by 53 ±â€¯2% and 51 ±â€¯7%, respectively, while Tn (5 and 50 µg/g) inhibited AChE activity by 35 ±â€¯4% and 80 ±â€¯9%, respectively (p < 0.05; n = 6). Moreover, CECA at concentrations of 5, 25, and 50 µg/g decreased the locomotor activity of the cockroaches, diminishing the distance travelled and increasing the frequency and duration of immobile episodes similarly to Tn (0.3 µg/g) (p < 0.05, n = 40, respectively). CECA (5, 25 and 50 µg/g) induced an increase in the leg grooming behavior, but not in the movement of antennae, similarly to the effect of Tn (0.3 µg/g). In addition, both CECA (50 µg/200 µl) and Tn (0.3 µg/200 µl) induced a negative chronotropism in the insect heart (37 ±â€¯1 and 47 ±â€¯8 beats/min in 30 min, respectively) (n = 9, p > 0.05). Finally, CECA (50 µg/g), Tn (0.3 µg/g) and neostigmine (50 µg/g) caused significant neuromuscular failure, as indicated by the monitoring of the in vivo neuromuscular function of the cockroaches, during 100 min (n = 6, p < 0.05, respectively). In conclusion, sublethal doses of CECA provoked entomotoxicity. The Tn-like effects of CECA on Nauphoeta cinerea cockroaches encompass both the central and peripheral nervous systems in our insect model. The inhibitory activity of CECA on AChE boosts a cascade of signaling events involving octopaminergic/dopaminergic neurotransmission. Therefore, this study indicates that this insect model could potentially be used as a powerful, practical, and inexpensive tool to understand the impacts of eutrophication and for orientating decontamination processes.

A high throughput targeted and non-targeted method for the analysis of microcystins and anatoxin-A using on-line solid phase extraction coupled to liquid chromatography-quadrupole time-of-flight high resolution mass spectrometry.

Microcystins are cyclic heptapeptide hepatotoxins produced by cyanobacteria in freshwater. Sample preparation for the analysis of these cyanotoxins in water from algal blooms can take up to several days due to the matrix complexity and the low detection limits required to comply with current legislation. Moreover, there is a large number of unknown microcystins that could potentially exist in the environment resulting from different amino acid substitutions into the microcystin skeletal structure. To tackle these problems, the present study involved the development of a high throughput method based on on-line solid phase extraction coupled to liquid chromatography that could provide quantitative results for 12 microcystin variants (LR, YR, RR, HtyR, HilR, WR, LW, LA, LF, LY, Dha7-LR, and Dha7-RR) and anatoxin-A in less than 3 h with detection limits between 0.004 and 0.01 µg L-1 and expanded uncertainty between 4 and 14%. Data-dependent acquisition was employed for the non-targeted analysis of these cyanotoxins. Filtering the data based on structure diagnostic fragments, two unknown microcystin variants not previously reported in the literature were detected. The structures Leu1-microcystin-Met(O)R and Leu1-microcystin-LY were fully characterized by accurate mass measurement, collision-induced dissociation, and fragmentation prediction software.

Toxicity at the Edge of Life: A Review on Cyanobacterial Toxins from Extreme Environments.

Cyanotoxins are secondary metabolites produced by cyanobacteria, of varied chemical nature and toxic effects. Although cyanobacteria thrive in all kinds of ecosystems on Earth even under very harsh conditions, current knowledge on cyanotoxin distribution is almost restricted to freshwaters from temperate latitudes. In this review, we bring to the forefront the presence of cyanotoxins in extreme environments. Cyanotoxins have been reported especially in polar deserts (both from the Arctic and Antarctica) and alkaline lakes, but also in hot deserts, hypersaline environments, and hot springs. Cyanotoxins detected in these ecosystems include neurotoxins-anatoxin-a, anatoxin-a (S), paralytic shellfish toxins, ß-methylaminopropionic acid, N-(2-aminoethyl) glycine and 2,4-diaminobutyric acid- and hepatotoxins -cylindrospermopsins, microcystins and nodularins-with microcystins being the most frequently reported. Toxin production there has been linked to at least eleven cyanobacterial genera yet only three of these (Arthrospira, Synechococcus and Oscillatoria) have been confirmed as producers in culture. Beyond a comprehensive analysis of cyanotoxin presence in each of the extreme environments, this review also identifies the main knowledge gaps to overcome (e.g., scarcity of isolates and -omics data, among others) toward an initial assessment of ecological and human health risks in these amazing ecosystems developing at the very edge of life.

Cyanobacterial Toxins of the Laurentian Great Lakes, Their Toxicological Effects, and Numerical Limits in Drinking Water.

Cyanobacteria are ubiquitous phototrophic bacteria that inhabit diverse environments across the planet. Seasonally, they dominate many eutrophic lakes impacted by excess nitrogen (N) and phosphorus (P) forming dense accumulations of biomass known as cyanobacterial harmful algal blooms or cyanoHABs. Their dominance in eutrophic lakes is attributed to a variety of unique adaptations including N and P concentrating mechanisms, N2 fixation, colony formation that inhibits predation, vertical movement via gas vesicles, and the production of toxic or otherwise bioactive molecules. While some of these molecules have been explored for their medicinal benefits, others are potent toxins harmful to humans, animals, and other wildlife known as cyanotoxins. In humans these cyanotoxins affect various tissues, including the liver, central and peripheral nervous system, kidneys, and reproductive organs among others. They induce acute effects at low doses in the parts-per-billion range and some are tumor promoters linked to chronic diseases such as liver and colorectal cancer. The occurrence of cyanoHABs and cyanotoxins in lakes presents challenges for maintaining safe recreational aquatic environments and the production of potable drinking water. CyanoHABs are a growing problem in the North American (Laurentian) Great Lakes basin. This review summarizes information on the occurrence of cyanoHABs in the Great Lakes, toxicological effects of cyanotoxins, and appropriate numerical limits on cyanotoxins in finished drinking water.

Efficient Enzymatic Routes for the Synthesis of New Eight-membered Cyclic ß-Amino Acid and ß-Lactam Enantiomers.

Efficient enzymatic resolutions are reported for the preparation of new eight-membered ring-fused enantiomeric ß-amino acids [(1R,2S)-9 and (1S,2R)-9] and ß-lactams [(1S,8R)-3, (1R,8S)-3 (1S,8R)-4 and (1R,8S)-7], through asymmetric acylation of (±)-4 (E > 100) or enantioselective hydrolysis (E > 200) of the corresponding inactivated (±)-3 or activated (±)-4 ß-lactams, catalyzed by PSIM or CAL-B in an organic solvent. CAL-B-catalyzed ring cleavage of (±)-6 (E > 200) resulted in the unreacted (1S,8R)-6, potential intermediate for the synthesis of enantiomeric anatoxin-a. The best strategies, in view of E, reaction rate and product yields, which underline the importance of substrate engineering, are highlighted.

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

BACKGROUND: Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. RESULTS: The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. CONCLUSION: Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress.

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg(-1) of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.

Uptake of the cyanobacterial neurotoxin, anatoxin-a, and alterations in oxidative stress in the submerged aquatic plant Ceratophyllum demersum.

The prevalence of cyanobacterial blooms in fresh water bodies worldwide has become a serious environmental problem. The blooms can increase the occurrence of cyanobacterial neurotoxin, anatoxin-a, and this toxin can interact with aquatic plants and other pivotal components of aquatic ecosystems. Despite this, several questions regarding the uptake of the toxin by aquatic plants and its association with toxic effects still remain. This study investigated the uptake of anatoxin-a in relation to alterations in oxidative stress, estimated by changes in lipid peroxidation and tocopherol contents (alpha-, beta-, gamma-, and delta-tocopherol), in the submerged aquatic plant, Ceratophylum demersum, at environmentally relevant concentrations. Exposure to five different concentrations of anatoxin-a (0.005, 0.05, 0.5, 5 and 50µgl(-1)) for 24h increased concentrations in C. demersum in a dose-dependent manner. All four forms of tocopherols were elevated at low concentrations of anatoxin-a (0.005. 0.05. 0.5 and 5µgl(-1)). However, a decline in the four tocopherol forms along with a high level of lipid peroxidation was observed at 50µgl(-1) exposure dose. During 336-h exposure to 15µgl(-1) anatoxin-a, rapid toxin uptake during the first 24h and subsequent steady accumulation of the toxin were observed. The four tocopherol forms increased in response to anatoxin-a uptake, attaining their maximum levels together with a significant increase of lipid peroxidation after 12 or 24h. After 24-h exposure, the four tocopherol forms decreased gradually without recovery. The results clearly indicate that anatoxin-a uptake can cause a disturbance of the oxidative stress in the aquatic plant, and depending on the concentration and exposure duration, oxidative damage occurs.

A cohesive Microcoleus strain cluster causes benthic cyanotoxic blooms in rivers worldwide.

Over the last two decades, proliferations of benthic cyanobacteria producing derivatives of anatoxin-a have been reported in rivers worldwide. Here, we follow up on such a toxigenic event happening in the Areuse river in Switzerland and investigate the diversity and genomics of major bloom-forming riverine benthic cyanobacteria. We show, using 16S rRNA-based community profiling, that benthic communities are dominated by Oscillatoriales. We correlate the detection of one Microcoleus sequence variant matching the Microcoleus anatoxicus species with the presence of anatoxin-a derivatives and use long-read metagenomics to assemble complete circular genomes of the strain. The main dihydro-anatoxin-a-producing strain in the Areuse is distinct from strains isolated in New Zealand, the USA, and Canada, but forms a monophyletic strain cluster with them with average nucleotide identity values close to the species threshold. Compared to the rest of the Microcoleus genus, the toxin-producing strains encode a 15 % smaller genome, lacking genes for the synthesis of some essential vitamins. Toxigenic mats harbor a distinct microbiome dominated by proteobacteria and bacteroidetes, which may support cyanobacterial growth by providing them with essential nutrients. We recommend that strains closely related to M. anatoxicus be monitored internationally in order to help predict and mitigate similar cyanotoxic events.

Inhibitory effects of toxic Dolichospermum flos-aquae and anatoxin-a on inducible defenses of Daphnia magna.

Cyanobacterial blooms, resulting from serious eutrophication, can produce various cyanotoxins and severely disrupt aquatic ecosystems. Inducible defenses are adaptive traits developed by prey in response to predation risks. However, the effects of the increasing proportion of cyanobacteria and cyanotoxins produced during cyanobacterial blooms on the inducible defenses of cladocerans, particularly in terms of behavioral defenses, remain unclear. In this study, we selected Daphnia magna and investigated the defensive traits against predation risks by the predator Rhodeus ocellatus under different ratios of cyanobacteria (Dolichospermum flos-aquae) and green algae (Scenedesmus obliquus), as well as varying concentrations of anatoxin-a (ATX), a cyanotoxin. We recorded the inducible defensive traits involving to morphology, behavior, and offspring production of D. magna. Results showed that the body length of D. magna at sexual maturity and the number of offspring in the first brood were significantly reduced by the presence of D. flos-aquae. Moreover, when the proportion of D. flos-aquae reached 75% and 100%, D. magna did not develop to sexual maturity. Furthermore, D. flos-aquae inhibited the formation of inducible behavioral defense of D. magna, with a stronger inhibitory effect as the proportion of D. flos-aquae increased. In this experiment, the effects of ATX on the morphological traits at sexual maturity and offspring production of D. magna were minor, but ATX still had the potential to inhibit the formation of inducible behavioral defense. We confirmed that changes in the proportion of cyanobacteria and green algae as well as the production of ATX by cyanobacteria during cyanobacterial blooms can affect the growth, development, and inducible defensive traits of cladocerans, potentially altering their population dynamics during such events.

Risk quick sketch: Soil captured most anatoxin-a and microcystin-RR rather than cylindrospermopsin and microcystin-LA/-LY.

Cyanobacteria proliferate in warm, nutrient-rich environments and release toxic secondary metabolites into natural waters. Using cyanotoxin-contaminated water to irrigate crops could expose humans and biota, but the risk may be low if agricultural soils can sorb and retain cyanotoxins. In this report, we compared the sorption and desorption capacities of multi-class cyanotoxins/anabaenopeptins in soils of variable properties with a batch sorption procedure. The target compounds were anabaenopeptin-A, anabaenopeptin-B, anatoxin-a, cylindrospermopsin, and microcystins -LR, -RR, -LA, -LY, -LW, and -LF. Based on solid-liquid distribution coefficients (Kd), we classified cylindrospermopsin and microcystin-LA/-LY as "very low sorptivity", anabaenopeptin-A, -B and microcystin-LR, -LF, and -LW as "low sorptivity", and anatoxin-a and microcystin-RR as "medium sorptivity". We remain concerned about irrigating agricultural land with water contaminated with high levels of CYN and MC-LA/-LY because of their relatively low affinity and high desorption proportion in soils. The results also suggest that soil sorption can be an effective immobilization pathway for anatoxin-a and microcystin-RR. The generated data will be useful for prioritizing research on the most bioavailable cyanotoxins/anabaenopeptins that are likely to be released by the soil matrix, for environmental risk assessment.

Analytical Methods for Anatoxin-a Determination: A Review.

Anatoxin-a (ATX-a) is a potent neurotoxin produced by several species of cyanobacteria whose exposure can have direct consequences, including neurological disorders and death. The increasing prevalence of harmful cyanobacterial blooms makes the detection and reliable assessment of ATX-a levels essential to prevent the risk associated with public health. Therefore, the aim of this review is to compile the analytical methods developed to date for the detection and quantification of ATX-a levels alone and in mixtures with other cyanotoxins and their suitability. A classification of the analytical methods available is fundamental to make an appropriate choice according to the type of sample, the equipment available, and the required sensitivity and specificity for each specific purpose. The most widely used detection technique for the quantification of this toxin is liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytical methods reviewed herein focus mainly on water and cyanobacterial samples, so the need for validated analytical methods in more complex matrices (vegetables and fish) for the determination of ATX-a to assess dietary exposure to this toxin is evidenced. There is currently a trend towards the validation of multitoxin methods as opposed to single-ATX-a determination methods, which corresponds to the real situation of cyanotoxins' confluence in nature.

Analyzing the neurotoxic effects of anatoxin-a and saxitoxin in zebrafish larvae.

Global warming due to climate change, as well as freshwater eutrophication caused by anthropogenic activities are responsible, among other factors, for an increasing occurrence of harmful algal blooms (HABs) in aquatic systems. These can lead to the generation of cyanotoxins, secondary metabolites coming from cyanobacteria, producing adverse effects in living organisms including death. This research aims to study the effects that two neurotoxins, anatoxin-a (ATX-a) and saxitoxin (STX), have on living organisms. Once the stability of both compounds in water was determined for a 24 h period using ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-MS/MS), zebrafish larvae were exposed to different levels of toxins (1 ng L-1, 10 ng L-1, 100 ng L-1 and 1 µg L-1) during 24 h. Behavioral studies including vibrational startle response (VSR), habituation to vibrational stimuli, basal locomotor activity (BLM) and visual motor response (VMR) were performed using Danio Vision system, and neurotransmitters (NTs) from 15-head pools of control and exposed zebrafish larvae were extracted and analyzed by UPLC-MS/MS. Both compounds induced hypolocomotion in the individuals, while 10 and 100 ng L-1 of ATX-a significantly increased methionine (120 % and 126 %, respectively) and glutamate levels (118 % and 129 %, respectively). Saxitoxin enhanced 3-metoxytyramine (3-MT) levels at 1 ng L-1 by 185 %. The findings of this study show that both studied cyanotoxins influence the behavior of zebrafish larvae as well as their metabolism.

Proteome changes in larval zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) exposed to (±) anatoxin-a.

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following  (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01-3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

A systematic review on guanitoxin: General characteristics and ecological risks.

Guanitoxin (GNT) is a potent cyanotoxin, with a relatively low number of publications (n = 51) compared to other cyanotoxins. Among the published studies, 35 % were on the effect of the toxin in animals, mainly in rodents and in vitro testing, followed by studies that identified species of cyanobacteria that produce GNT in aquatic systems and consequently accidental poisoning in wild and domestic animals (27 %). Studies that developed or tested methods for identifying the molecule, based on colorimetric and analytical techniques, represented 14 %, while 8 % were on GNT biosynthesis. Review articles and chemical isolation (6 %) and on the stability of the molecule (4 %) were the topics with the lowest number of publications. The results show the occurrence of GNT was identified mainly in eutrophic environments with a higher incidence in the American continent. Chemical characteristics of the molecule, such as short half-life in the environment, instability in solutions with alkaline pH values, temperature >23 °C, added to the lack of an analytical standard, are factors that make it difficult to identify and quantify it. However, GNT monitoring can be performed using LC-MS-MRM methods or genes specific to the newly discovered molecule.