Target Mechanisms of the Cyanotoxin Cylindrospermopsin in Immortalized Human Airway Epithelial Cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o-) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.

The cyanotoxin cylindrospermopsin slows down cell cycle progression and extends metaphase duration in immortalised human airway epithelial cells.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs worldwide in aquatic environments. It is known for its delayed effects upon oral uptake in animals and humans. A less well studied route of CYN internalisation is the inhalation of CYN-contaminated aerosols. We analyzed potential effects of different CYN concentrations (1, 2.5 and 5 µmol/l) on cultures of immortalised human bronchial epithelial cells (16HBE14o-). Impedance, a proxi for cell attachment to the culture support, cell spreading, cell growth and cell proliferation, was measured using an Acea iCELLigence device. Cell division rate and metaphase duration were determined using time lapse movies (Nikon Biostation II) of CYN-exposed cell cultures. Western blot studies were used to determine expression levels of cell cycle regulator proteins, the cyclins B1, D1 and A2. Our investigations revealed that exposure of cells to CYN concentrations of 1 µmol/l or higher led to a concentration- and time-dependent attenuation of impedance development as well as cell proliferation rate, and an extension of the metaphase of the cell cycle. CYN-mediated downregulation of cyclins B1 and D1 may be part of the underlying cell physiological mechanism. These results indicate that exposure of airways in humans and animals to aerosolised CYN over longer periods may be harmful.

Immunomodulatory Effects of Pure Cylindrospermopsin in Rats Orally Exposed for 28 Days.

Cylindrospermopsin (CYN) is a ubiquitous cyanotoxin showing increasing incidence worldwide. CYN has been classified as a cytotoxin and, among its toxic effects, its immunotoxicity is scarcely studied. This work investigates for the first time the influence of oral CYN exposure (18.75; 37.5 and 75 µg/kg b.w./day, for 28 days) on the mRNA expression of selected interleukin (IL) genes (IL-1ß, IL-2, IL-6, Tumor Necrosis Factor alpha (TNF-α), Interferon gamma (IFN-γ)) in the thymus and the spleen of male and female rats, by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, their serum levels were also measured by a multiplex-bead-based immunoassay, and a histopathological study was performed. CYN produced immunomodulation mainly in the thymus of rats exposed to 75 µg CYN/kg b.w./day in both sexes. However, in the spleen only IL-1ß and IL-2 (males), and TNF-α and IFN-γ (females) expression was modified after CYN exposure. Only female rats exposed to 18.75 µg CYN/kg b.w./day showed a significant decrease in TNF-α serum levels. There were no significant differences in the weight or histopathology in the organs studied. Further research is needed to obtain a deeper view of the molecular mechanisms involved in CYN immunotoxicity and its consequences on long-term exposures.

The Toxicity Testing of Cyanobacterial Toxins In vivo and In vitro by Mouse Bioassay: A Review.

Different biological methods based on bioactivity are available to detect cyanotoxins, including neurotoxicity, immunological interactions, hepatotoxicity, cytotoxicity, and enzymatic activity. The mouse bioassay is the first test employed in laboratory cultures, cell extracts, and water bloom materials to detect toxins. It is also used as a traditional method to estimate the LD50. Concerning the ease of access and low cost, it is the most common method for this purpose. In this method, a sample is injected intraperitoneally into adult mice, and accordingly, they are assayed and monitored for about 24 hours for toxic symptoms. The toxin can be detected using this method from minutes to a few hours; its type, e.g., hepatotoxin, neurotoxin, etc., can also be determined. However, this method is nonspecific, fails to detect low amounts, and cannot distinguish between homologues. Although the mouse bioassay is gradually replaced with new chemical and immunological methods, it is still the main technique to detect the bioactivity and efficacy of cyanotoxins using LD50 determined based on the survival time of animals exposed to the toxin. In addition, some countries oppose animal use in toxicity studies. However, high cost, ethical considerations, low-sensitivity, non-specificity, and prolonged processes persuade researchers to employ chemical and functional analysis techniques. The qualitative and quantitative analyses, as well as high specificity and sensitivity, are among the advantages of cytotoxicity tests to investigate cyanotoxins. The present study aimed at reviewing the results obtained from in vitro and in vivo investigations of the mouse bioassay to detect cyanotoxins, including microcystins, cylindrospermopsin, saxitoxins, etc.

Cyanotoxins and Food Contamination in Developing Countries: Review of Their Types, Toxicity, Analysis, Occurrence and Mitigation Strategies.

Cyanotoxins have gained global public interest due to their potential to bioaccumulate in food, which threatens human health. Bloom formation is usually enhanced under Mediterranean, subtropical and tropical climates which are the dominant climate types in developing countries. In this context, we present an up-to-date overview of cyanotoxins (types, toxic effects, analysis, occurrence, and mitigation) with a special focus on their contamination in (sea)food from all the developing countries in Africa, Asia, and Latin America as this has received less attention. A total of 65 publications have been found (from 2000 until October 2021) reporting the contamination by one or more cyanotoxins in seafood and edible plants (five papers). Only Brazil and China conducted more research on cyanotoxin contamination in food in comparison to other countries. The majority of research focused on the detection of microcystins using different analytical methods. The detected levels mostly surpassed the provisional tolerable daily intake limit set by the World Health Organization, indicating a real risk to the exposed population. Assessment of cyanotoxin contamination in foods from developing countries still requires further investigations by conducting more survey studies, especially the simultaneous detection of multiple categories of cyanotoxins in food.

BMAA, Methylmercury, and Mechanisms of Neurodegeneration in Dolphins: A Natural Model of Toxin Exposure.

Dolphins are well-regarded sentinels for toxin exposure and can bioaccumulate a cyanotoxin called ß-N-methylamino-l-alanine (BMAA) that has been linked to human neurodegenerative disease. The same dolphins also possessed hallmarks of Alzheimer's disease (AD), suggesting a possible association between toxin exposure and neuropathology. However, the mechanisms of neurodegeneration in dolphins and the impact cyanotoxins have on these processes are unknown. Here, we evaluate BMAA exposure by investigating transcription signatures using PCR for dolphin genes homologous to those implicated in AD and related dementias: APP, PSEN1, PSEN2, MAPT, GRN, TARDBP, and C9orf72. Immunohistochemistry and Sevier Münger silver staining were used to validate neuropathology. Methylmercury (MeHg), a synergistic neurotoxicant with BMAA, was also measured using PT-GC-AFS. We report that dolphins have up to a three-fold increase in gene transcription related to Aß+ plaques, neurofibrillary tangles, neuritic plaques, and TDP-43+ intracytoplasmic inclusions. The upregulation of gene transcription in our dolphin cohort paralleled increasing BMAA concentration. In addition, dolphins with BMAA exposures equivalent to those reported in AD patients displayed up to a 14-fold increase in AD-type neuropathology. MeHg was detected (0.16-0.41 µg/g) and toxicity associated with exposure was also observed in the brain. These results demonstrate that dolphins develop neuropathology associated with AD and exposure to BMAA and MeHg may augment these processes.

Immunotoxic Effects Induced by Microcystins and Cylindrospermopsin: A Review.

Cyanotoxin occurrence is gaining importance due to anthropogenic activities, climate change and eutrophication. Among them, Microcystins (MCs) and Cylindrospermopsin (CYN) are the most frequently studied due to their ubiquity and toxicity. Although MCs are primary classified as hepatotoxins and CYN as a cytotoxin, they have been shown to induce deleterious effects in a wide range of organs. However, their effects on the immune system are as yet scarcely investigated. Thus, to know the impact of cyanotoxins on the immune system, due to its importance in organisms' homeostasis, is considered of interest. A review of the scientific literature dealing with the immunotoxicity of MCs and CYN has been performed, and both in vitro and in vivo studies have been considered. Results have confirmed the scarcity of reports on the topic, particularly for CYN. Decreased cell viability, apoptosis or altered functions of immune cells, and changed levels and mRNA expression of cytokines are among the most common effects reported. Underlying mechanisms, however, are still not yet fully elucidated. Further research is needed in order to have a full picture of cyanotoxin immunotoxicity.

Cylindrospermopsin impairs vascular smooth muscle cells by P53-mediated apoptosis due to ROS overproduction.

Cylindrospermopsin (CYN) is a toxic secondary metabolite from cyanobacteria that can cause cardiovascular disease. However, the study of CYN-induced cardiovascular toxicity in vitro is very limited and the mechanism is remain to be clarified. Vascular smooth muscle cells (VMSCs) have an important function in maintaining the structural and functional integrity of the aortic wall, and are an important in vitro model for cardiovascular research. Thus, the effects of CYN exposure (2, 20, 200, and 2000 nM) on VMSCs were analyzed. In vitro study, results showed that CYN exposure decreased VMSCs viability, inhibited VMSCs migration, induced DNA damage, destroyed cytoskeleton, changed cell morphology, promoted VMSCs apoptosis, and increased intracellular reactive oxygen species (ROS) levels. In addition, CYN could induce the activities of SOD, CAT and GPX, and promote the expressions of SOD1, CAT, GPx1, p53 and Bax genes and inhibit the expression of Bcl-2 gene, leading to a higher ratio of Bax/Bcl-2. Taken together, CYN may induce ROS overproduction, leading to increased p53 expression and ultimately promoting VSMC apoptosis. Therefore, the present study demonstrates that CYN could impair VMSCs, leading to vascular developmental defects and angiocardiopathy.

Cyanotoxins and the Nervous System.

Cyanobacteria are capable of producing a wide range of bioactive compounds with many considered to be toxins. Although there are a number of toxicological outcomes with respect to cyanobacterial exposure, this review aims to examine those which affect the central nervous system (CNS) or have neurotoxicological properties. Such exposures can be acute or chronic, and we detail issues concerning CNS entry, detection and remediation. Exposure can occur through a variety of media but, increasingly, exposure through air via inhalation may have greater significance and requires further investigation. Even though cyanobacterial toxins have traditionally been classified based on their primary mode of toxicity, increasing evidence suggests that some also possess neurotoxic properties and include known cyanotoxins and unknown compounds. Furthermore, chronic long-term exposure to these compounds is increasingly being identified as adversely affecting human health.

Cysteine biosynthesis contributes to ß-methylamino-l-alanine tolerance in Escherichia coli.

In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin ß-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 µg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H2O2 in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.

Is Exposure to BMAA a Risk Factor for Neurodegenerative Diseases? A Response to a Critical Review of the BMAA Hypothesis.

In a literature survey, Chernoff et al. (2017) dismissed the hypothesis that chronic exposure to ß-N-methylamino-L-alanine (BMAA) may be a risk factor for progressive neurodegenerative disease. They question the growing scientific literature that suggests the following: (1) BMAA exposure causes ALS/PDC among the indigenous Chamorro people of Guam; (2) Guamanian ALS/PDC shares clinical and neuropathological features with Alzheimer's disease, Parkinson's disease, and ALS; (3) one possible mechanism for protein misfolds is misincorporation of BMAA into proteins as a substitute for L-serine; and (4) chronic exposure to BMAA through diet or environmental exposures to cyanobacterial blooms can cause neurodegenerative disease. We here identify multiple errors in their critique including the following: (1) their review selectively cites the published literature; (2) the authors reported favorably on HILIC methods of BMAA detection while the literature shows significant matrix effects and peak coelution in HILIC that may prevent detection and quantification of BMAA in cyanobacteria; (3) the authors build alternative arguments to the BMAA hypothesis, rather than explain the published literature which, to date, has been unable to refute the BMAA hypothesis; and (4) the authors erroneously attribute methods to incorrect studies, indicative of a failure to carefully consider all relevant publications. The lack of attention to BMAA research begins with the review's title which incorrectly refers to BMAA as a "non-essential" amino acid. Research regarding chronic exposure to BMAA as a cause of human neurodegenerative diseases is emerging and requires additional resources, validation, and research. Here, we propose strategies for improvement in the execution and reporting of analytical methods and the need for additional and well-executed inter-lab comparisons for BMAA quantitation. We emphasize the need for optimization and validation of analytical methods to ensure that they are fit-for-purpose. Although there remain gaps in the literature, an increasingly large body of data from multiple independent labs using orthogonal methods provides increasing evidence that chronic exposure to BMAA may be a risk factor for neurological illness.

BMAA, Neurodegeneration, and Neuroprotection.

In this volume, studies springing from a BMAA symposium held in Salt Lake City, Utah, in April 2019 are presented. Although most studies of neurotoxicity consider the effects of BMAA as an isolated molecule, it is now known that environmental exposures can be to a combination of BMAA-related molecules, including enantiomers, isomers, other co-occurring cyanotoxins, and BMAA carbamates. Within the body, BMAA may exist in equilibrium with α- and ß-carbamates formed in the presence of bicarbonate. BMAA and its isomers 2,4-DAB and AEG, accumulate over decades in biocrusts and persist at depths in soil profiles of the Gulf deserts. In Florida, releases of cyanobacterially ladened water from Lake Okeechobee can extend into coastal environments where diatoms and possibly dinoflagellates also produce BMAA and isomers in addition to brevetoxins. Along the African Lake Chad, neurotoxic risks from consumption of dried cyanobacterial cakes may, however, be outweighed by their amino acid addition to otherwise protein-deficient diets. Discrepancies in the detection and quantification of BMAA from different laboratories likely originate in the use of different analytical methods. C-18 columns, used to study derivatized BMAA, can efficiently separate BMAA from its isomers in validated methods, while validation is not possible for HILIC columns in the study of underivatized BMAA, since they do not adequately separate BMAA from its isomer BAMA. The presence of BMAA dimers, metal adducts, and carbamates may result in underestimation of BMAA by mass spectrometry. BMAA research led to the identification of the dietary amino acid L-serine as a neuroprotective molecule. In animal and clinical trials, L-serine appears to slow neurodegeneration, although the modes of action are still under study. Based on zebra fish sensitivity to platinum-based chemotherapeutic agents, investigators have found that L-serine reduces reactive oxygen species (ROS) but does not protect auditory hybridoma cells from cisplatin. Another possible mode of action of L-serine, induction of autophagic-lysosomal enzymes, is also being explored. The hypothesis that cyanobacterial exposures in general, and chronic exposures to BMAA in particular, may prove to be risk factors for neurodegenerative illnesses has not been without critics. Emerging from the symposium, a multi-authored response to one such critical paper appears in this collection of articles. Instead of waiting until there is a conclusive proof of risk, the adoption of the "precautionary default principle," proposed by Ingvar Brandt and his colleagues in Sweden, is suggested. Avoidance of exposures to cyanobacterial blooms and other sources of BMAA is suggested, until further research indicates such precautions to be unnecessary.