Microcystin-LR in drinking water: An emerging role of mitochondrial-induced epigenetic modifications and possible mitigation strategies.

Algal blooms are a serious menace to freshwater bodies all over the world. These blooms typically comprise cyanobacterial outgrowths that produce a heptapeptide toxin, Microcystin-LR (MC-LR). Chronic MC-LR exposure impairs mitochondrial-nuclear crosstalk, ROS generation, activation of DNA damage repair pathways, apoptosis, and calcium homeostasis by interfering with PC/MAPK/RTK/PI3K signaling. The discovery of the toxin's biosynthesis pathways paved the way for the development of molecular techniques for the early detection of microcystin. Phosphatase inhibition-based bioassays, high-performance liquid chromatography, and enzyme-linked immunosorbent tests have recently been employed to identify MC-LR in aquatic ecosystems. Biosensors are an exciting alternative for effective on-site analysis and field-based characterization. Here, we present a synthesis of evidence supporting MC-LR as a mitotoxicant, examine various detection methods, and propose a novel theory for the relevance of MC-LR-induced breakdown of mitochondrial machinery and its myriad biological ramifications in human health and disease.

A probabilistic hazard assessment for cyanobacterial toxins accounting for regional geography and water body trophic status.

Under climate change scenarios freshwater eutrophication is expected to increase, and with it the occurrence of cyanobacterial toxin-producing harmful algal blooms. In the current study, microcystin toxin occurrence data from literature sources and a long-term provincial monitoring program were used to conduct a probabilistic hazard assessment for Alberta, Canada. The large temporal and spatial range of data makes Alberta a model system for identifying regional geography and water body trophic status factors driving toxin concentrations. Environmental exposure distributions of microcystin concentrations were plotted and used to identify the likelihood of a given sample exceeding water guideline values as a function of regional geography, total phosphorus and chlorophyll-a concentration. This process identified regions with intensive cultivation and those most prone to water deficits associated with climate change to be most associated with exceedances of regulatory guideline values. Elevated phosphorus and chlorophyll-a concentrations were also drivers of toxin occurrence. This assessment can be used to identify water bodies of greatest risk to human and animal populations from cyanotoxins and thereby inform regulators as to most effective monitoring strategies.

Draft genome sequences of three Planktothrix rubescens strains cultivated from a eutrophic lake in Norwalk, Ohio (USA).

Draft genomes were generated for three filamentous toxin-producing cyanobacterial strains cultivated from aquatic sources in Ohio sequenced by NovaSeq S4. Here, we report the classification and genome statistics of Planktothrix rubescens PR221, PR222, and PR223.

Systemic inflammatory response to daily exposure to microcystin-LR and the underlying gut microbial mechanisms.

Cyanobacterial toxins have raised global concerns due to potential chronic disease implications from daily drinking water exposure, which remain largely unknown despite extensive research on their acute effects. To understand the mechanisms underlying microcystin-LR (MC-LR)-induced inflammation-associated diseases. Mice were exposed to MC-LR for one year at concentrations comparable to human environmental exposure levels. Comprehensive pathological observation and multi-omics approaches based on 16S rRNA gene sequencing, untargeted metabolomics, transcriptomics and proteomics were conducted across various organs. Daily exposure to MC-LR induced intestinal microbial dysbiosis and colitis-like changes. It also caused systemic chronic inflammation marked by elevated serum levels of inflammatory cytokines, inflammation-associated pathological changes, and identification of infection-related genes/proteins within the gut-brain-spleen-liver axis. Furthermore, multi-omics analysis across organs suggested that Muribaculaceae may promote a systemic infection-inflammatory response, relying on kynurenine metabolites signaling in peripheral tissues. In contrast, Lachnospiraceae may act an opposing role, dependent on intestinal indole derivatives via the neuroimmunomodulation pathway. A fecal microbiota transplantation experiment confirmed that alterations in Muribaculaceae and Lachnospiraceae resulting from exposure to MC-LR triggered the local and systemic chronic inflammation in mice. This study light on the potential strategies employed by gut microbial community in regulating MC-induced inflammation-associated chronic diseases under repeated exposure through drinking water.

Unprecedented Harmful algal bloom in the UK and Ireland's largest lake associated with gastrointestinal bacteria, microcystins and anabaenopeptins presenting an environmental and public health risk.

Harmful Algal Blooms (HABs) are outbreaks of aquatic toxic microalgae emerging as a global problem driven by nutrient enrichment, global climate change and invasive species. We uniquely describe a HAB of unprecedented duration, extent and magnitude during 2023 in Lough Neagh; the UK and Ireland's largest freshwater lake, using an unparalleled combination of satellite imagery, nutrient analysis, 16S rRNA gene sequencing and cyanotoxin profiling. The causative agent Microcystis aeruginosa accounted for over a third of DNA in water samples though common bacterioplankton species also bloomed. Water phosphate levels were hypertrophic and drove local algal biomass. The HAB pervaded the entire ecosystem with algal mats accumulating around jetties, marinas and lock gates. Over 80 % of bacterial DNA isolated from algal mat samples consisted of species associated with wildfowl or livestock faeces and human-effluent wastewater including 13 potential pathogens that can cause serious human illness including: E. coli, Salmonella, Enterobacter and Clostridium among others. Ten microcystins, nodularin and two anabaenopeptin toxins were confirmed as present (with a further microcystin and four anabaenopeptins suspected), with MC-RR and -LR in high concentrations at some locations (1,137-18,493 µg/L) with MC-LR exceeding World Health Organisation (WHO) recreational exposure guidelines in all algal mats sampled. This is the first detection of anabaenopeptins in any waterbody on the island of Ireland. Notwithstanding the ecological impacts, this HAB represented an environmental and public health risk, curtailing recreational activities in-and-around the lake and damaging local businesses. Reducing agricultural runoff and discharge from human-effluent wastewater treatment to manage nutrient loading, and the public health risk, should be the top priority of stakeholders, especially government. Key recommendations include Nature-based Solutions that avoid conflict with the productivity and profitability of the farming sector enhancing sustainability. We hope this stimulates real-world action to resolve the problems besetting this internationally important ecosystem.

Increased Prorenin Expression in the Kidneys May Be Involved in the Abnormal Renal Function Caused by Prolonged Environmental Exposure to Microcystin-LR.

Toxic algae in eutrophic lakes produce cyanotoxic microcystins. Prior research on the effect of microcystin-LR in the kidney utilized intraperitoneal injections, which did not reflect natural exposure. Oral microcystin-LR research has focused on renal function and histopathology without examining the molecular mechanisms. The present study aimed to evaluate the mechanism of microcystin-LR in the kidneys via oral administration in WKAH/HkmSlc rats over 7 weeks, alongside stimulation of the proximal tubular cells. Although there were no differences in the concentrations of plasma albumin, blood urea nitrogen, and creatinine, which are parameters of renal function, between the control and microcystin-LR-administrated rats, prorenin expression was significantly increased in the renal cortex of the rats administered microcystin-LR and the microcystin-LR-treated proximal tubular cells. The expression levels of (pro)renin receptor (PRR), transforming growth factor-ß1 (TGFß1), and α-smooth muscle actin (α-SMA) in the renal cortex did not differ significantly between the control and microcystin-LR-administered rats. However, the expression levels of prorenin were significantly positively correlated with those of PRR, TGFß1, and α-SMA in the renal cortex of rats administered microcystin-LR. Additionally, a significant positive correlation was observed between the expression levels of TGFß1 and α-SMA. Collectively, increased prorenin expression caused by the long-term consumption of microcystin-LR may initiate a process that influences renal fibrosis and abnormal renal function by regulating the expression levels of PRR, TGFß1, and α-SMA.

Community Structure and Toxicity Potential of Cyanobacteria during Summer and Winter in a Temperate-Zone Lake Susceptible to Phytoplankton Blooms.

Cyanobacterial blooms are increasingly common during winters, especially when they are mild. The goal of this study was to determine the summer and winter phytoplankton community structure, cyanotoxin presence, and toxigenicity in a eutrophic lake susceptible to cyanobacterial blooms throughout the year, using classical microscopy, an analysis of toxic cyanometabolites, and an analysis of genes involved in biosynthesis of cyanotoxins. We also assessed whether cyanobacterial diversity in the studied lake has changed compared to what was reported in previous reports conducted several years ago. Moreover, the bloom-forming cyanobacterial strains were isolated from the lake and screened for cyanotoxin presence and toxigenicity. Cyanobacteria were the main component of the phytoplankton community in both sampling times, and, in particular, Oscillatoriales were predominant in both summer (Planktothrix/Limnothrix) and winter (Limnothrix) sampling. Compared to the winter community, the summer community was denser; richer in species; and contained alien and invasive Nostocales, including Sphaerospermopsis aphanizomenoides, Raphidiopsis raciborskii, and Raphidiopsis mediterranea. In both sampling times, the blooms contained toxigenic species with genetic determinants for the production of cylindrospermopsin and microcystins. Toxicological screening revealed the presence of microcystins in the lake in summer but no cyanotoxins in the winter period of sampling. However, several cyanobacterial strains isolated from the lake during winter and summer produced anabaenopeptins and microcystins. This study indicates that summer and winter blooms of cyanobacteria in the temperate zone can differ in biomass, structure, and toxicity, and that the toxic hazards associated with cyanobacterial blooms may potentially exist during winter.

First report of PST-producing Microseira wollei from China reveals its novel toxin profile.

Microseira wollei, a globally distributed freshwater bloom-forming benthic cyanobacterium, is known for its production of cyanotoxins and taste and odor (T&O). While CYN (Cylindrospermopsin)-producing populations of M. wollei are confined to Australia, PST (Paralytic shellfish toxins)-producing populations have been exclusively documented in North America. In this study, four benthic cyanobacterial strains, isolated from West Lake in China, were identified as M. wollei based on morphological and phylogenetic analyses. Detection of sxtA gene and UPLC-MS/MS analysis conclusively confirmed the PST-producing capability of M. wollei CHAB5998. In the phylogenetic tree of 16S rDNA, M. wollei strains formed a monophyletic group with two subclades. Notably, non-PST-producing Chinese strains clustered with Australian strains in Clade II, while all other strains, including PST-producing ones, clustered in Clade I. Additionally, CHAB5998 contains ten PST variants, of which STX, NEO, GTX2, GTX3, GTX5 and C1 were identified for the first time in M. wollei. Sequence analysis of PST biosynthetic gene cluster (sxt) genes indicated potential base variations, gene rearrangements, insertions, and deletions in the strain CHAB5998. Also, sxt gene has a longer evolutionary history in M. wollei than that in cyanobacteria from Nostocales. Multiple recombination breakpoints detected in sxt genes and the inconsistency in the topology of the phylogenetic trees between sxt and 16S rDNA suggested that multiple horizontal gene transfers (HGT) have occurred. Overall, the present study marks the first documented occurrence of PST-producing M. wollei outside of North America and identifies it as the first toxic freshwater benthic cyanobacterium in China. This revelation implies that benthic cyanobacteria may pose a higher environmental risk in China than previously acknowledged.

Toxicological effects of nodularin on the reproductive endocrine system of female zebrafish (Danio rerio).

Nodularin is a potent cyanotoxin that has been detected in aquatic environments as well as in the body of aquatic organisms throughout the world, but its effects on the reproductive system are yet to be explored. The present study investigated the toxic effects of environmentally relevant concentrations of nodularin on the reproductive endocrine system of female zebrafish (Danio rerio). After exposure to nodularin for 14 days, decreased gonadosomatic Index (GSI), germinal vesicle breakdown (GVBD), and decreased level of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17ß-estradiol (E2) level and increased testosterone (T) content in female zebrafish suggested that nodularin may disrupt both oocyte growth and maturation. In support of this data, alteration in different marker gene expression on the hypothalamic-pituitary-gonadal-liver (HPGL) axis was observed. Transcriptional levels of genes related to steroidogenesis including cytochrome P450 aromatase (cyp19a1a) in the ovary and primary vitellogenin genes (vtg1, vtg2, and vtg3) in the liver were down-regulated and marker genes for oxidative stress (sod, cat, and gpx) were up-regulated on HPGL axis. These findings revealed for the first time that nodularin is a potent endocrine-disrupting compound posing oxidative stress and causes reproductive endocrine toxicity in female zebrafish, emphasizing the importance of assessing its environmental risks.

Microcystin removal by microbial communities from a coastal lagoon: Influence of abiotic factors, bacterioplankton composition and estimated functions.

Toxic cyanobacterial blooms present a substantial risk to public health due to the production of secondary metabolites, notably microcystins (MCs). Microcystin-LR (MC-LR) is the most prevalent and toxic variant in freshwater. MCs resist conventional water treatment methods, persistently impacting water quality. This study focused on an oligohaline shallow lagoon historically affected by MC-producing cyanobacteria, aiming to identify bacteria capable of degrading MC and investigating the influence of environmental factors on this process. While isolated strains did not exhibit MC degradation, microbial assemblages directly sourced from lagoon water removed MC-LR within seven days at 25 ºC and pH 8.0. The associated bacterial community demonstrated an increased abundance of bacterial taxa assigned to Methylophilales, and also Rhodospirillales and Rhodocyclales to a lesser extent. However, elevated atmospheric temperatures (45 ºC) and acidification (pH 5.0 and 3.0) hindered MC-LR removal, indicating that extreme environmental changes could contribute to prolonged MC persistence in the water column. This study highlights the importance of considering environmental conditions in order to develop strategies to mitigate cyanotoxin contamination in aquatic ecosystems.

Microcystin-leucine arginine induces the proliferation of cholangiocytes and cholangiocarcinoma cells through the activation of the Wnt/ß-catenin signaling pathway.

Background: Microcystin-leucine arginine (MC-LR) is a cyanobacterial hepatotoxic toxin found in water sources worldwide, including in northeastern Thailand, where opisthorchiasis-associated cholangiocarcinoma (CCA) is most prevalent. MC-LR is a potential carcinogen; however, its involvement in liver fluke-associated CCA remains ambiguous. Here, we aimed to evaluate the effect of MC-LR on the progression of CCA via the Wnt/ß-catenin pathway in vitro. Methods: Cell division, migration, cell cycle transition, and MC-LR transporter expression were evaluated in vitro through MTT assay, wound healing assay, flow cytometry, and immunofluorescence staining, respectively. Following a 24-h treatment of cultured cells with 1, 10, 100, and 1,000 nM of MC-LR, the proliferative effect of MC-LR on the Wnt/ß-catenin signaling pathway was investigated using immunoblotting and qRT-PCR analysis. Immunohistochemistry was used to determine ß-catenin expression in CCA tissue compared to adjacent tissue. Results: Human immortalized cholangiocyte cells (MMNK-1) and a human cell line established from opisthorchiasis-associated CCA (KKU-213B) expressed the MC-LR transporter and internalized MC-LR. Exposure to 10 nM and 100 nM of MC-LR notably enhanced cells division and migration in both cell lines (P < 0.05) and markedly elevated the percentage of S phase cells (P < 0.05). MC-LR elevated PP2A expression by activating the Wnt/ß-catenin signaling pathway and suppressing phosphatase activity. Inhibition of the ß-catenin destruction complex genes (Axin1 and APC) led to the upregulation of ß-catenin and its downstream target genes (Cyclin D1 and c-Jun). Inhibition of Wnt/ß-catenin signaling by MSAB confirmed these results. Additionally, ß-catenin was significantly expressed in cancerous tissue compared to adjacent areas (P < 0.001). Conclusions: Our findings suggest that MC-LR promotes cell proliferation and progression of CCA through Wnt/ß-catenin pathway. Further evaluation using invivo experiments is needed to confirm this observation. This finding could promote health awareness regarding MC-LR intake and risk of CCA.

Comprehensive analysis of cyanobacterial secondary metabolites distribution and toxicity in urban water bodies.

This study addresses the increasing concern regarding cyanotoxin contamination of water bodies, highlighting the diversity of these toxins and their potential health implications. Cyanobacteria, which are prevalent in aquatic environments, produce toxic metabolites, raising concerns regarding human exposure and associated health risks, including a potential increase in cancer risk. Although existing research has primarily focused on well-known cyanotoxins, recent technological advancements have revealed numerous unknown cyanotoxins, necessitating a comprehensive assessment of multiple toxin categories. To enhance the cyanotoxin databases, we optimized the CyanoMetDB cyanobacterial secondary metabolites database by incorporating secondary fragmentation patterns using the Mass Frontier fragmentation data prediction software. Water samples from diverse locations in Shanghai were analyzed using high-resolution mass spectrometry. Subsequently, the toxicity of cyanobacterial metabolites in the water samples was examined through acute toxicity assays using the crustacean Thamnocephalus platyurus. After 24 h of exposure, the semi-lethal concentrations (LC50) of the water samples ranged from 0.31 mg L-1 to 1.78 mg L-1 (MC-LR equivalent concentration). Our findings revealed a critical correlation between the overall concentration of cyanobacterial metabolites and toxicity. The robust framework and insights of this study underscore the need for an inclusive approach to water quality management, emphasizing continuous efforts to refine detection methods and comprehend the broader ecological impact of cyanobacterial blooms on aquatic ecosystems.

Microbial Diversity Impacts Non-Protein Amino Acid Production in Cyanobacterial Bloom Cultures Collected from Lake Winnipeg.

Lake Winnipeg in Manitoba, Canada is heavily impacted by harmful algal blooms that contain non-protein amino acids (NPAAs) produced by cyanobacteria: N-(2-aminoethyl)glycine (AEG), ß-aminomethyl-L-alanine (BAMA), ß-N-methylamino-L-alanine (BMAA), and 2,4-diaminobutyric acid (DAB). Our objective was to investigate the impact of microbial diversity on NPAA production by cyanobacteria using semi-purified crude cyanobacterial cultures established from field samples collected by the Lake Winnipeg Research Consortium between 2016 and 2021. NPAAs were detected and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using validated analytical methods, while Shannon and Simpson alpha diversity scores were determined from 16S rRNA metagenomic sequences. Alpha diversity in isolate cultures was significantly decreased compared to crude cyanobacterial cultures (p < 0.001), indicating successful semi-purification. BMAA and AEG concentrations were higher in crude compared to isolate cultures (p < 0.0001), and AEG concentrations were correlated to the alpha diversity in cultures (r = 0.554; p < 0.0001). BAMA concentrations were increased in isolate cultures (p < 0.05), while DAB concentrations were similar in crude and isolate cultures. These results demonstrate that microbial community complexity impacts NPAA production by cyanobacteria and related organisms.

The synchronicity of bloom-forming cyanobacteria transcription patterns and hydrogen peroxide dynamics.

Hydrogen peroxide is a reactive oxygen species (ROS) naturally occurring at low levels in aquatic environments and production varies widely across different ecosystems. Oxygenic photosynthesis generates hydrogen peroxide as a byproduct, of which some portion can be released to ambient water. However, few studies have examined hydrogen peroxide dynamics in relation to cyanobacterial harmful algal blooms (cHABs). A year-long investigation of algal succession and hydrogen peroxide dynamics was conducted at the Caloosahatchee River, Florida, USA. We aimed to identify potential biological mechanisms responsible for elevated hydrogen peroxide production during cHAB events through the exploration of the freshwater microbial metatranscriptome. Hydrogen peroxide concentrations were elevated from February to September of 2021 when cyanobacteria were active and abundant. We observed one Microcystis cHAB event in spring and one in winter. Both had distinct nutrient uptake and cyanotoxin gene expression patterns. While meaningful levels of microcystin were only detected during periods of elevated hydrogen peroxide, cyanopeptolin was by far the most expressed cyanotoxin during the spring bloom when hydrogen peroxide was at its yearly maxima. Gene expressions of five microbial enzymes (Rubisco, superoxide dismutase, cytochrome b559, pyruvate oxidase, and NADH dehydrogenase) positively correlated to hydrogen peroxide concentrations. Additionally, there was higher nitrogen-fixing gene (nifDKH) expression by filamentous cyanobacteria after the spring bloom but no secondary bloom formation occurred. Overall, elevated environmental hydrogen peroxide concentrations were linked to cyanobacterial dominance and greater expression of specific enzymes in the photosynthesis of cyanobacteria. This implicates cyanobacterial photosynthesis and growth results in increased hydrogen peroxide generation as reflected in measured environmental concentrations.

Two duckweed species exhibit variable tolerance to microcystin-LR exposure across genotypic lineages.

Cyanotoxins produced by harmful cyanobacteria blooms can damage freshwater ecosystems and threaten human health. Floating macrophytes may be used as a means of biocontrol by limiting light and resources available to cyanobacteria. However, genetic variation in macrophyte sensitivity to cyanotoxins could influence their suitability as biocontrol agents. We investigated the influence of such intraspecific variation on the response of two rapidly growing duckweed species, Lemna minor and Spirodela polyrhiza, often used in nutrient and metal bioremediation. We assessed two biomarkers related to productivity (biomass and chlorophyll A production) and two related to fitness measures (population size and growth rate). Fifteen genetic lineages of each species were grown in media containing common cyanotoxin microcystin-LR at ecologically relevant concentrations or control media for a period of twelve days. Genotype identity had a strong impact on all biomarker responses. Microcystin concentration slightly increased the final population sizes of both macrophyte species with a marginal effect on growth rate of L. minor and the chlorophyll A production of S. polyrhiza, but overall these species were very tolerant of microcystin. The strong tolerance supports the potential use of these plants as bioremediators of cyanobacterial blooms. However, differential impact of microcystin exposure discovered in single lineage models among genotypes indicates a potential for cyanotoxins to act as selective forces, necessitating attention to genotype selection for bioremediation.

What, How, When, and Where: Spatiotemporal Water Quality Hazards of Cyanotoxins in Subtropical Eutrophic Reservoirs.

Though toxins produced during harmful blooms of cyanobacteria present diverse risks to public health and the environment, surface water quality surveillance of cyanobacterial toxins is inconsistent, spatiotemporally limited, and routinely relies on ELISA kits to estimate total microcystins (MCs) in surface waters. Here, we employed liquid chromatography tandem mass spectrometry to examine common cyanotoxins, including five microcystins, three anatoxins, nodularin, cylindrospermopsin, and saxitoxin in 20 subtropical reservoirs spatially distributed across a pronounced annual rainfall gradient. Probabilistic environmental hazard analyses identified whether water quality values for cyanotoxins were exceeded and if these exceedances varied spatiotemporally. MC-LR was the most common congener detected, but it was not consistently observed with other toxins, including MC-YR, which was detected at the highest concentrations during spring with many observations above the California human recreation guideline (800 ng/L). Cylindrospermopsin was also quantitated in 40% of eutrophic reservoirs; these detections did not exceed a US Environmental Protection Agency swimming/advisory level (15,000 ng/L). Our observations have implications for routine water quality monitoring practices, which traditionally use ELISA kits to estimate MC levels and often limit collection of surface samples during summer months near reservoir impoundments, and further indicate that spatiotemporal surveillance efforts are necessary to understand cyanotoxins risks when harmful cyanobacteria blooms occur throughout the year.

Nanoparticles for Mitigation of Harmful Cyanobacterial Blooms.

With the rapid advancement of nanotechnology and its widespread applications, increasing amounts of manufactured and natural nanoparticles (NPs) have been tested for their potential utilization in treating harmful cyanobacterial blooms (HCBs). NPs can be used as a photocatalyst, algaecide, adsorbent, flocculant, or coagulant. The primary mechanisms explored for NPs to mitigate HCBs include photocatalysis, metal ion-induced cytotoxicity, physical disruption of the cell membrane, light-shielding, flocculation/coagulation/sedimentation of cyanobacterial cells, and the removal of phosphorus (P) and cyanotoxins from bloom water by adsorption. As an emerging and promising chemical/physical approach for HCB mitigation, versatile NP-based technologies offer great advantages, such as being environmentally benign, cost-effective, highly efficient, recyclable, and adaptable. The challenges we face include cost reduction, scalability, and impacts on non-target species co-inhabiting in the same environment. Further efforts are required to scale up to real-world operations through developing more efficient, recoverable, reusable, and deployable NP-based lattices or materials that are adaptable to bloom events in different water bodies of different sizes, such as reservoirs, lakes, rivers, and marine environments.

Blue Green Algae.

Blue green algae cyanotoxins have become increasingly more prevalent due to environmental, industrial, and agricultural changes that promote their growth into harmful algal blooms. Animals are usually exposed via water used for drinking or bathing, though specific cases related to equines are very limited. The toxic dose for horses has not been determined, and currently only experimental data in other animals can be relied upon to aid in case interpretation and treatment. Treatment is mostly limited to supportive care, and preventative control methods to limit exposures are more likely to aid in animal health until more research has been performed.

Cyanobacterial anatoxin-a does not induce in vitro developmental neurotoxicity, but changes gene expression patterns in co-exposure with all-trans retinoic acid.

Cyanobacterial blooms are increasing in frequency and intensity globally, and impacting recreational waters as well as waters used for drinking water provisioning. They are sources of bioactive metabolites including retinoids and the neurotoxin anatoxin-a. Here, we investigated the effects of anatoxin-a on a differentiating in vitro human neural stem cell model previously characterised with retinoic acids. Effects on protein and gene expression upon exposure for 9 or 18 days to anatoxin-a alone or in co-exposure with all-trans retinoic acid were evaluated using a panel of neural and glial differentiation biomarkers. Anatoxin-a did not cause distinct developmental neurotoxicity alone, or in co-exposure with retinoic acid. However, in line with its excitotoxicity, in co-exposure with 200 nM all-trans retinoic acid it reduced the differentiation of acetylcholinergic neuron subtypes in the culture at 1000 nM (highest tested concentration). While this could have substantial functional implications for the developing nervous system, there is no indication for developmental neurotoxicity beyond its (excito-)toxicity to acetylcholinergic neurons, which only occurred in co-exposure to all-trans retinoic acid.

A rapid and efficient method using electroporation for releasing intracellular microcystin toxins from cultured and naturally occurring cyanobacterial cells in lake water.

In cyanotoxin measurements, effective release of intracellular cyanotoxins through cell lysis is pivotal. The conventional method for cell lysis is repeated freeze-thaw (F-T), which has several disadvantages, including poor reproducibility since it is operator and equipment dependency and time-consuming. In this study, a rapid and sensitive method was developed using irreversible electroporation, reducing quantification time by over 6 h compared to F-T. Focusing on microcystins (MCs), we developed the most optimal electroporation medium (50 mM Tris (pH 7.0) with 0.5 % SDS) and determined the optimal intensity of electroporation using Microcystis culture. Microcystis cell rupture was validated by scanning electron microscopy. COMSOL simulations mirrored experimental conditions. Compared to F-T, this new method generated an average 13.7 % (6.7 ppb) more MCs from lake water samples (p ≥ 0.05). This innovation, surpassing the time-consuming F-T process, emerges as a valuable tool for timely decision-making in water safety advisory and cyanotoxin management in various settings.