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Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk+/− cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN−), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN−. Furthermore, the reactive oxygen species (ROS) increased with time (0–24 h) and CYN concentration (0–1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN−, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN−, which highlights the importance of studying toxic features in their natural scenario.

Cylindrospermopsin (CYN) is a cyanotoxin with an increasing occurrence, and therefore it is important to elucidate its toxicity profile. CYN has been classified as a cytotoxin, although the scientific literature has already revealed that it affects a wide range of organs and systems. However, research on its potential immunotoxicity is still limited. Thus, this study aimed to evaluate the impact of CYN on two human cell lines representative of the immune system: THP-1 (monocytes) and Jurkat (lymphocytes). CYN reduced cell viability, leading to mean effective concentrations (EC50 24 h) of 6.00 ± 1.04 µM and 5.20 ± 1.20 µM for THP-1 and Jurkat cells, respectively, and induced cell death mainly by apoptosis in both experimental models. Moreover, CYN decreased the differentiation of monocytes to macrophages after 48 h of exposure. In addition, an up-regulation of the mRNA expression of different cytokines, such as interleukin (IL) 2, IL-8, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ), was also observed mainly after 24 h exposure in both cell lines. However, only an increase in TNF-α in THP-1 supernatants was observed by ELISA. Overall, these results suggest the immunomodulatory activity of CYN in vitro. Therefore, further research is required to evaluate the impact of CYN on the human immune system.

Microcystins (MCs) and cylindrospermopsin (CYN), although classified as hepatotoxins and cytotoxins, respectively, have been shown to also induce toxic effects in many other systems and organs. Among them, their potential endocrine disruption (ED) activity has been scarcely investigated. Considering the increasing relevance of ED on humans, mammals, and aquatic organisms, this work aimed to review the state-of-the-art regarding the toxic effects of MCs and CYN at this level. It has been evidenced that MCs have been more extensively investigated than CYN. Reported results are contradictory, with the presence or absence of effects, but experimental conditions also vary to a great extent. In general, both toxins have shown ED activity mediated by very different mechanisms, such as estrogenic responses via a binding estrogen receptor (ER), pathological changes in several organs and cells (testis, ovarian cells), and a decreased gonad-somatic index. Moreover, toxic effects mediated by reactive oxygen species (ROS), changes in transcriptional responses on several endocrine axes and steroidogenesis-related genes, and changes in hormone levels have also been reported. Further research is required in a risk assessment frame because official protocols for assessment of endocrine disrupters have not been used. Moreover, the use of advanced techniques would aid in deciphering cyanotoxins dose-response relationships in relation to their ED potential.

Cyanotoxins are secondary metabolites produced by different types of cyanobacteria. Among them, Cylindrospermopsin (CYN) and Microcystins (MCs) stand out due to their wide geographical distribution and toxicity in various organs, including the kidney, which is involved in their distribution and elimination. However, the renal toxicity caused by CYN and MCs has hardly been studied. The aim of this work was to assess the cytotoxicity effects caused by CYN and MC-LR in the renal cell line HEK293, and for the first time, the influence of CYN on the gene expression of selected genes in these cells by quantitative real-time PCR (qRT-PCR). CYN caused an upregulation in the gene expression after exposure to the highest concentration (5 µg/mL) and the longest time of exposure (24 h). Moreover, shotgun proteomic analysis was used to assess the molecular responses of HEK293 cells after exposure to the individuals and combinations of CYN + MC-LR. The simultaneous exposure to both cyanotoxins caused a greater number of alterations in protein expression compared to single toxins, causing changes in the cellular, lipid and protein metabolism and in protein synthesis and transport. Further studies are needed to complete the toxicity molecular mechanisms of both CYN and MC-LR at the renal level.

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-1β, 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-1β 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.

Cylindrospermopsin (CYN) is a cyanotoxin whose incidence has been increasing in the last decades. Due to its capacity to exert damage at different levels of the organism, it is considered a cytotoxin. Although the main target organ is the liver, recent studies indicate that CYN has potential toxic effects on the nervous system, both in vitro and in vivo. Thus, the aim of the present work was to study the effects of this cyanotoxin on neuronal viability and synaptic integrity in murine primary cultures of neurons exposed to environmentally relevant concentrations (0–1 µg/mL CYN) for 12, 24, and 48 h. The results demonstrate a concentration- and time-dependent decrease in cell viability; no cytotoxicity was detected after exposure to the cyanotoxin for 12 h, while all of the concentrations assayed decreased this parameter after 48 h. Furthermore, CYN was also demonstrated to exert damage at the synaptic level in a murine primary neuronal culture in a concentration- and time-dependent manner. These data highlight the importance of studying the neurotoxic properties of this cyanotoxin in different experimental models.

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.

Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

Toxic cyanobacterial blooms are a major contaminant in inland aquatic ecosystems. Furthermore, toxic blooms are carried downstream by rivers and waterways to estuarine and coastal ecosystems. Concerning marine and estuarine animal species, very little is known about how these species are affected by the exposure to freshwater cyanobacteria and cyanotoxins. So far, most of the knowledge has been gathered from freshwater bivalve molluscs. This work aimed to infer the sensitivity of the marine mussel Mytilus galloprovincialis to single as well as mixed toxic cyanobacterial cultures and the underlying molecular responses mediated by toxic cyanobacteria. For this purpose, a mussel exposure experiment was outlined with two toxic cyanobacteria species, Microcystis aeruginosa and Chrysosporum ovalisporum at 1 × 105 cells/mL, resembling a natural cyanobacteria bloom. The estimated amount of toxins produced by M. aeruginosa and C. ovalisporum were respectively 0.023 pg/cell of microcystin-LR (MC-LR) and 7.854 pg/cell of cylindrospermopsin (CYN). After 15 days of exposure to single and mixed cyanobacteria, a depuration phase followed, during which mussels were fed only non-toxic microalga Parachlorella kessleri. The results showed that the marine mussel is able to filter toxic cyanobacteria at a rate equal or higher than the non-toxic microalga P. kessleri. Filtration rates observed after 15 days of feeding toxic microalgae were 1773.04 mL/ind.h (for M. aeruginosa), 2151.83 mL/ind.h (for C. ovalisporum), 1673.29 mL/ind.h (for the mixture of the 2 cyanobacteria) and 2539.25 mL/ind.h (for the non-toxic P. kessleri). Filtering toxic microalgae in combination resulted in the accumulation of 14.17 ng/g dw MC-LR and 92.08 ng/g dw CYN. Other physiological and biochemical endpoints (dry weight, byssus production, total protein and glycogen) measured in this work did not change significantly in the groups exposed to toxic cyanobacteria with regard to control group, suggesting that mussels were not affected with the toxic microalgae. Nevertheless, proteomics revealed changes in metabolism of mussels related to diet, specially evident in those fed on combined cyanobacteria. Changes in metabolic pathways related with protein folding and stabilization, cytoskeleton structure, and gene transcription/translation were observed after exposure and feeding toxic cyanobacteria. These changes occur in vital metabolic processes and may contribute to protect mussels from toxic effects of the toxins MC-LR and CYN.

Cyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins.

The cyanobacterial toxin Cylindrospermopsin (CYN), a potent protein synthesis inhibitor, is increasingly being found in freshwater bodies infested by cyanobacterial blooms worldwide. Moreover, it has been reported to be implicated in human intoxications and animal mortality. Recently, the alteration of the activity and gene expression of some glutathione related enzymes in tilapias (Oreochromis niloticus) exposed to a single dose of CYN has been reported. However, little is known about the effects induced by repeated doses of this toxin in tilapias exposed by immersion and the potential reversion of these biochemical alterations after two different depuration periods (3 or 7 days). In the present study, tilapias were exposed by immersion to repeated doses of a CYN-containing culture of Aphanizomenon ovalisporum during 14 days, and then were subjected to depuration periods (3 or 7 days) in clean water in order to examine the potential reversion of the effects observed. The activity and relative mRNA expression by real-time polymerase chain reaction (PCR) of the antioxidant enzymes glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST), and also the sGST protein abundance by Western blot analysis were evaluated in liver and kidney of fish. Results showed significant alterations in most of the parameters evaluated and their recovery after 3 days (GPx activity, sGST relative abundance) or 7 days (GPx gene expression, sGST activity). These findings not only confirm the oxidative stress effects produced in fish by cyanobacterial cells containing CYN, but also show the effectiveness of depuration processes in mitigating the CYN-containing culture toxic effects.

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria (i.e., Aphanizomenon ovalisporum). CYN is a tricyclic alkaloid combined with a guanidine moiety. It is well known that CYN inhibits both protein and glutathione synthesis, and also induces genotoxicity and the alteration of different oxidative stress biomarkers. Although the liver and kidney appear to be the main target organs for this toxin based on previous studies, CYN also affects other organs. In the present study, we studied the distribution of CYN in fish (Oreochromis niloticus) under two different exposure scenarios using immunohistochemical (IHC) techniques. In the first method, fish were exposed acutely by intraperitoneal injection or by gavage to 200 µg pure CYN/Kg body weight (bw), and euthanized after 24 h or five days of exposure. In the second method, fish were exposed by immersion to lyophilized A. ovalisporum CYN-producing cells using two concentration levels (10 or 100 µg/L) for two different exposure times (7 or 14 days). The IHC was carried out in liver, kidney, intestine, and gills of fish. Results demonstrated a similar pattern of CYN distribution in both experimental methods. The organ that presented the most immunopositive results was the liver, followed by the kidney, intestine, and gills. Moreover, the immunolabeling signal intensified with increasing time in both assays, confirming the delayed toxicity of CYN, and also with the increment of the dose, as it is shown in the sub-chronic assay. Thus, IHC is shown to be a valuable technique to study CYN distribution in these organisms.

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04−2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.

Reports on the occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) have increased worldwide because of CYN toxic effects in humans and animals. If contaminated waters are used for plant irrigation, these could represent a possible CYN exposure route for humans. For the first time, a method employing solid phase extraction and quantification by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) of CYN was optimized in vegetables matrices such as lettuce (Lactuca sativa). The validated method showed a linear range, from 5 to 500 ng CYN g−1 of fresh weight (f.w.), and detection and quantitation limits (LOD and LOQ) of 0.22 and 0.42 ng CYN g−1 f.w., respectively. The mean recoveries ranged between 85 and 104%, and the intermediate precision from 12.7 to 14.7%. The method showed to be robust for the three different variables tested. Moreover, it was successfully applied to quantify CYN in edible lettuce leaves exposed to CYN-contaminated water (10 µg L−1), showing that the tolerable daily intake (TDI) in the case of CYN could be exceeded in elderly high consumers. The validated method showed good results in terms of sensitivity, precision, accuracy, and robustness for CYN determination in leaf vegetables such as lettuce. More studies are needed in order to prevent the risks associated with the consumption of CYN-contaminated vegetables.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that is gaining importance, owing to its increasing expansion worldwide and the increased frequency of its blooms. CYN mainly targets the liver, but also involves other organs. Various mechanisms have been associated with its toxicity, such as protein synthesis inhibition, oxidative stress, etc. However, its toxic effects are not yet fully elucidated and additional data for hazard characterization purposes are required. In this regard, in vitro methods can play an important role, owing to their advantages in comparison to in vivo trials. The aim of this work was to compile and evaluate the in vitro data dealing with CYN available in the scientific literature, focusing on its toxicokinetics and its main toxicity mechanisms. This analysis would be useful to identify research needs and data gaps in order to complete knowledge about the toxicity profile of CYN. For example, it has been shown that research on various aspects, such as new emerging toxicity effects, the toxicity of analogs, or the potential interaction of CYN with other cyanotoxins, among others, is still very scarce. New in vitro studies are therefore welcome.