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The occurrence of toxic blooms of cyanobacteria has been a matter of public health interest due to the cyanotoxins produced by these microorganisms. Cylindrospermopsin (CYN) is a cyanotoxin of particular concern due to its toxic effects on humans. This study investigated the removal and effects of CYN in ripened slow sand filters (SSFs) treating water from Paranoá Lake, Brasilia, Brazil. Four pilot-scale SSFs were ripened and operated for 74 days. Two contamination peaks with CYN were applied along the filtration run. The improvement of any of the evaluated water quality parameters was not affected by the presence of CYN in the raw water. The SSFs efficiently removed CYN, presenting concentrations lower than 0.8 µg/L in the filtered water. The microbiota of the SSFs were dominated by protozoa of the genus Euglypha and amoebas of the genera Arcella, Centropyxis, and Amoeba, together with some groups of rotifers. These microorganisms played a crucial role in removing total coliforms and E. coli. In addition, CYN was not identified as a determining factor in the microbiota composition.

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The cyanotoxin cylindrospermopsin (CYN) has become a significant environmental and human health concern due to its high toxicological potential and widespread distribution. High concentrations of cyanotoxins may be produced during cyanobacterial blooms. Special attention is required when these blooms occur in sources of water intended for human consumption since extracellular cyanotoxins are not effectively removed by conventional water treatments, leading to the need for advanced water treatment technologies such as the Fenton process to produce safe water. Thus, the present study aimed to investigate the application of the Fenton process for the degradation of CYN at bench-scale. The oxidation of CYN was evaluated by Fenton reaction at H2O2/Fe(II) molar ratio in a range of 0.4 to 4.0, with the highest degradation of about 81% at molar ratio of 0.4. Doubling the concentrations of reactants for the optimized H2O2/Fe(II) molar ratio, the CYN degradation efficiency reached 91%. Under the conditions studied, CYN degradation by the Fenton process followed a pseudo-first-order kinetic model with an apparent constant rate ranging from 0.813 × 10-3 to 1.879 × 10-3 s-1.

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Cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. Studies have demonstrated that the kidney is the most affected organ. CYN exposure leads to low-molecular-weight proteinuria and increased excretions of the tubular enzymes in mice, suggesting the damage caused by CYN is mainly tubular. However, the mechanism involved in CYN nephrotoxicity remains unknown. Thus, in order to evaluate the effects of CYN exposure (0.1, 0.5 and 1.0 µg/mL) on tubular renal cells LLC-PK1 distinct mechanisms were analyzed by assessing cell death using flow cytometry, albumin uptake by fluorescence analysis, Na+/K+-ATPase activity by a colorimetric method, RT-qPCR of genes related to tubular transport and function as well as internalization of CYN by ELISA. In this study, CYN was found to induce necrosis in all concentrations. CYN also decreased albumin uptake as well as downregulated megalin and dab2 expression, both proteins involved in albumin endocytosis process. Moreover, CYN appears to be internalized by renal tubular cells through a receptor-mediated endocytosis. Finally, the present study demonstrates that CYN is responsible for disrupting tubular cell transport and function in LLC-PK1 cells.

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The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN's lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

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RESUMO

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue

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Cyanobacterial metabolites are increasingly studied, in regards to their biosynthesis, ecological role, toxicity, and potential biomedical applications. However, the history of cyanotoxins prior to the last few decades is virtually unknown. Only a few paleolimnological studies have been undertaken to date, and these have focused exclusively on microcystins and cylindrospermopsins, both successfully identified in lake sediments up to 200 and 4700 years old, respectively. In this paper, we review direct extraction, quantification, and application of cyanotoxins in sediment cores, and put forward future research prospects in this field. Cyanobacterial toxin research is also compared to other paleo-cyanobacteria tools, such as sedimentary pigments, akinetes, and ancient DNA isolation, to identify the role of each tool in reproducing the history of cyanobacteria. Such investigations may also be beneficial for further elucidation of the biological role of cyanotoxins, particularly if coupled with analyses of other abiotic and biotic sedimentary features. In addition, we identify current limitations as well as future directions for applications in the field of paleolimnological studies on cyanotoxins.

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Human hepatoma cells (HepG2) were exposed to purified cylindrospermopsin (CYN), a potent toxicant for eukaryotic cells produced by several cyanobacteria. Exposure to 10 µg l-1 of CYN for 24 h resulted in alteration of expression of 48 proteins, from which 26 were identified through mass spectrometry. Exposure to 100 µg l-1 of CYN for 24 h affected nuclear area and actin filaments intensity, which can be associated with cell proliferation and toxicity. The proteins are implicated in different biological processes: protein folding, xenobiotic efflux, antioxidant defense, energy metabolism and cell anabolism, cell signaling, tumorigenic potential, and cytoskeleton structure. Protein profile indicates that CYN exposure may lead to alteration of glucose metabolism that can be associated with the supply of useful energy to cells respond to chemical stress and proliferate. Increase of G protein-coupled receptors (GPCRs), heterogeneous nuclear ribonucleoproteins (hnRNP), and reactive oxygen species (ROS) levels observed in HepG2 cells can associate with cell proliferation and resistance. Increase of MRP3 and glutathione peroxidase can protect cells against some chemicals and ROS. CYN exposure also led to alteration of the expression of cytoskeleton proteins, which may be associated with cell proliferation and toxicity.

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The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.

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The cyanobacterial toxin cylindrospermopsin (CYN) is of great concern in aquatic environments because of its incidence, multiple toxicity endpoints, and, therefore, the severity of health implications. It may bioaccumulate in aquatic food webs, resulting in high exposure concentrations to higher-order trophic levels, particularly humans. Because of accumulation at primary levels resulting from exposure to trace amounts of toxin, a sensitive analytical technique with proven aquatic applications is required. In the present study, a hydrophilic interaction liquid chromatographic-tandem mass spectrometric method with a lower limit of detection of 200 fg on column (signal-to-noise ratio = 3, n = 9) and a lower limit of quantification of 1 pg on column (signal-to-noise ratio = 11, n = 9) with demonstrated application in 4 aquatic organisms is described. The analytical method was optimized and validated with a linear range (r(2) = 0.999) from 0.1 ng mL(-1) to 100 ng mL(-1) CYN. Mean recovery of the extraction method was 98 ± 2%. Application of the method was demonstrated by quantifying CYN uptake in Scenedesmus subspicatus (green algae), Egeria densa (Brazilian waterweed), Daphnia magna (water flea), and Lumbriculus variegatus (blackworm) after 24 h of static exposure to 50 µg L(-1) CYN. Uptake ranged from 0.05% to 0.11% of the nominal CYN exposure amount. This constitutes a sensitive and reproducible method for extraction and quantification of unconjugated CYN with demonstrated application in 4 aquatic organisms, which can be used in further aquatic toxicological investigations.

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The cyanotoxin cylindrospermopsin (CYN) has lately been reported with a notorious toxicity to mammals. LASSBio-596 is a compound with anti-inflammatory actions. We aimed at evaluating the therapeutic effects of LASSBio-596 in a model of CYN-induced lung injury. Protocol #1: BALB/c mice received intratracheally (i.t.) 50-µL of saline or semi-purified extract of CYN (70 µg/kg). 18 h later, animals that received saline were gavaged with saline (SALSAL) or 50 mg/kg of LASSBio-596 (SALLAS), and mice that received CYN were gavaged with either saline (TOXSAL) or 50 mg/kg of LASSBio-596 (TOXLAS). Pulmonary mechanics was measured 6 h after gavage. Lungs were prepared for histology and inflammatory mediators determination. Protocol #2: Mice received 50-µL of CYN (70 µg/kg, i.t.) and 18 h later were gavaged with saline (NOT TREATED), or 50 mg/kg of LASSBio-596 (TREATED). Survival rates and pulmonary mechanics of the survivors were assessed. CYN exposure increased mechanical components, alveolar collapse, PMN cells and fiber deposition in the lungs, as well as the production of IL-1ß, IL-6 and KC in Protocol #1. LASSBio-596 attenuated those changes. TREATED mice in Protocol #2 presented significantly higher survival rates and tended to improve lung mechanics. Briefly, LASSBio-596 showed positive effects in mice exposed to CYN.

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Cylindrospermopsin (CYN) induces toxicity in pregnant mice when administered intraperitoneally. This study investigated whether oral exposure to CYN (0.03, 0.3 and 3 µg/kg) during pregnancy causes toxic effects and impairs gestation in rats. The results of reproductive performance and teratology studies were similar between the control and experimental dams. Our findings suggest that CYN consumption within the guideline values for drinking water is not able to promote foetal toxicity or alterations in rat reproductive performance.

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