Toxicity Of Cylindrospermopsin Research Articles

Ferrate(VI) mediated degradation of the potent cyanotoxin, cylindrospermopsin: Kinetics, products, and toxicity

The presence of cylindrospermopsin (CYN), a potent cyanotoxin, in drinking water sources poses a tremendous risk to humans and the environment. Detailed kinetic studies herein demonstrate ferrate(VI) (FeVIO42−, Fe(VI)) mediated oxidation of CYN and the model compound 6-hydroxymethyl uracil (6-HOMU) lead to their effective degradation under neutral and alkaline solution pH. A transformation product analysis indicated oxidation of the uracil ring, which has functionality critical to the toxicity of CYN. The oxidative cleavage of the C5=C6 double bond resulted in fragmentation of the uracil ring. Amide hydrolysis is a contributing pathway leading to the fragmentation of the uracil ring. Under extended treatment, hydrolysis, and extensive oxidation lead to complete destruction of the uracil ring skeleton, resulting in the generation of a variety of products including nontoxic cylindrospermopsic acid. The ELISA biological activity of the CYN product mixtures produced during Fe(VI) treatment parallels the concentration of CYN. These results suggest the products do not possess ELISA biological activity at the concentrations produced during treatment. The Fe(VI) mediated degradation was also effective in the presence of humic acid and unaffected by the presence of common inorganic ions under our experimental conditions. The Fe(VI) remediation of CYN and uracil based toxins appears a promising drinking water treatment process.

Combined methods elucidate the multi-organ toxicity of cylindrospermopsin (CYN) on Daphnia magna

Global water bodies are now at risk from inevitable cyanobacterial blooms and their production of multiple cyanotoxins, in particular cylindrospermopsin (CYN). However, research on the CYN toxicity and its molecular mechanisms is still limited, whilst the responses of aquatic species against CYN are uncovered. By integrating behavioral observations, chemical detections and transcriptome analysis, this study demonstrated that CYN exerted multi-organ toxicity to model species, Daphnia magna. The present study confirmed that CYN could cause protein inhibition by undermining total protein contents, and altered the gene expression related to proteolysis. Meantime, CYN induced oxidative stress by increasing reactive oxygen species (ROS) level, decreasing the glutathione (GSH) concentration, and interfered with protoheme formation process molecularly. Neurotoxicity led by CYN was solidly determined by abnormal swimming patterns, reduced acetylcholinesterase (AChE), and downward expression of muscarinic acetylcholine receptor (CHRM). Importantly, for the first time, this research determined CYN directly interfered with energy metabolism in cladocerans. CYN distinctively reduced filtration and ingestion rate by targeting on heart and thoracic limbs, which declined the energy intake, and could be further displayed by the reduction of motional strength and the trypsin concentration. These phenotypic alterations were supported by transcriptomic profile, including the down-regulation of oxidative phosphorylation and ATP synthesis. Moreover, CYN was speculated to trigger the self-defense responses of D. magna, known as “abandon-ship” by moderating lipid metabolism and distribution. This study, overall, comprehensively demonstrated the CYN toxicity and the responses of D. magna against it, which is of great significance to the advancements of CYN toxicity knowledge.

Cylindrospermopsin induces oocyte maturation and disrupts gene expression in zebrafish ovarian follicles

There is evidence that cylindrospermopsin (CYN) exerts reproductive toxicity in mice. However, little information is available concerning the toxicity of CYN in nonmammalian vertebrates. Here, we investigated the direct action of CYN on female reproduction by studying germinal vesicle breakdown, transcript abundance, caspase-3 activity, and testosterone production using cultured follicle-enclosed zebrafish oocytes as a model system. Treatment of follicles with 1,000 μg/L CYN significantly increased GVBD, Caspase-3 activity, and hCG-induced testosterone secretion. Exposure to CYN also reduced the abundance of 3βhsd as well as hCG-induced fshr and era transcripts and increased cyp19a1 mRNA levels. In summary, this study provides a framework for a better understanding of the adverse action of CYN on female reproduction in zebrafish and other vertebrate species. The findings are also relevant to developing valid biomarkers for CYN by measuring zebrafish oocyte maturation and gene expression.

Cylindrospermopsin directly disrupts spermatogenesis in isolated male zebrafish testis

Cylindrospermopsin (CYN) is a cytotoxin, and its documented effects in mammals include damage to several organs. CYN also has hormone-disrupting properties, including estrogenic activity, progesterone production inhibition, and apoptosis induction. While CYN has been reported to exert reproductive toxicity in mice, little is known about its effect on fish reproductive function. Using ex vivo organ culture, we investigated the direct action of CYN on the male reproductive system. Isolated zebrafish testis was exposed to 250, 500, and 1000 µg/L CYN for 24 h and 7 d, followed by histo-morphological analysis. The results demonstrate that exposure to CYN led to a decrease in cell types from all three phases of spermatogenesis in zebrafish testis. There were also significant changes in fshr, lhr, and igf3 transcript levels, as well as testosterone secretion following exposure to CYN. In summary, this study provides novel information on the adverse effects of CYN on testicular spermatogenesis and male reproduction in zebrafish. These results provide a framework for a better understanding of CYN toxicity and the mechanism underlying the adverse action of CYN on male reproduction in fish.

First occurrence of Cylindrospermopsin in the Azores (Lake São Brás, S. Miguel Island)

Anthropogenic impacts have been reported as one of the main causes of eutrophication in freshwater systems and nutrient availability, mainly from discharges, are key to cyanobacteria development and harmful cyanobacteria blooms (cyanoHABs) upsurge. As these scenarios are common in the Azores, a cyanobacteria monitoring program was implemented in the lakes of the archipelago, which comprises cyanobacteria identification and cyanotoxin (only microcystin) analysis in over 15 lakes. However other cyanotoxins should be searched as is the case of cylindrospermopsin (CYN). This cyanotoxin has been reported more recently in a wider range of countries, because of global warming. Using ESI–LC–MS/MS (electrospray ionization–liquid chromatography–mass spectrometry), the presence of CYN was investigated in samples from four eutrophic lakes. Cylindrospermopsin was detected in one sample from Lake Sao Bras, which represents a threat to environmental and public health. This is the first CYN identification in the Azores reinforcing the necessity for lake monitoring. We propose the use of a polyphasic approach to fully understand CYN presence in the Azorean lakes, investigating the producing taxa, the environmental factors that promote cyanobacteria growth, and CYN toxicity effects.

New Data on Cylindrospermopsin Toxicity.

Cylindrospermopsin (CYN) is a widely spread cyanotoxin that can occur in fresh water and food. This research aims to investigate CYN toxicity by studying the effects of drinking 0.25 nM of CYN-contaminated water from a natural source, and of the direct application of moderate concentrations of CYN on different animal targets. The chosen structures and activities are rat mitochondria inner membrane permeability, mitochondrial ATP synthase (ATPase) and rat liver diamine oxidase (DAO) activities (EC 1.4.3.22.), the force of the contraction of an excised frog heart preparation with functional innervation, and the viability of a human intestinal epithelial cell line (HIEC-6). The oral exposure to CYN decreased the reverse (hydrolase) activity of rat liver ATPase whereas its short-term, in vitro application was without significant effect on this organelle, DAO activity, heart contractions, and their neuronal regulation. The application of CYN reduced HIEC-6 cells’ viability dose dependently. It was concluded that CYN is moderately toxic for the human intestinal epithelial cells, where the regeneration of the epithelial layer can be suppressed by CYN. This result suggests that CYN may provoke pathological changes in the human gastrointestinal tract.

Uptake and Effects of Cylindrospermopsin: Biochemical, Physiological and Biometric Responses in The Submerged Macrophyte Egeria densa Planch

Cylindrospermopsin (CYN) is being detected in surface waters more commonly and frequently worldwide. This stable, extracellular cyanotoxin causes protein synthesis inhibition, thus posing a risk to aquatic biota, including macrophytes, which serve as primary producers. Nevertheless, data regarding the effects caused by environmental concentrations of CYN is still limited. In the presented study, the uptake of CYN at environmental concentrations by the submerged macrophyte Egeria densa was investigated. Bioaccumulation, changes in the plant biomass, as well as shoot-length were assessed as responses. Variations in the cellular H2O2 levels, antioxidative enzyme activities, as well as concentrations and ratios of the photosynthetic pigments were also measured. E. densa removed 54% of CYN within 24 h and up to 68% after 336 h; however, CYN was not bioaccumulated. The antioxidative enzyme system was activated by CYN exposure. Pigment concentrations decreased with exposure but normalized after 168 h. The chlorophyll a to b ratio increased but normalized quickly thereafter. Carotenoids and the ratio of carotenoids to total chlorophylls increased after 96 h suggesting participation in the antioxidative system. Growth stimulation was observed. The ability to remove CYN and resistance to CYN toxicity within 14 days proved E. densa as suitable for phytoremediation; nonetheless, prolonged exposure (32 days) resulted in adverse effects related to CYN uptake, which needs to be studied further.

Probing high-affinity aptamer binding region and development of aptasensor platform for the detection of cylindrospermopsin

Cylindrospermopsin (CYN) is one of the most concerning cyanotoxins due to its potential toxicity and spreading to various environments including drinking water. CYN has potential interferences with human and animal metabolic pathways, which influence the functions of organs including liver, kidneys, lungs, etc. CYN is involved in the inhibition of protein synthesis and detachment of ribosomes from the endoplasmic reticulum membrane. It also interacts with soluble proteins, which are associated with protein translations. It is believed that cytochrome 450 is responsible for the rapid toxicity of CYN. Researchers are urged to develop a high-throughput screening method for the detection of CYN in water. Construction of low cost, rapid, and sensitive analytical methods for the detection of CYN is challenging. Here, we used graphene oxide (GO) as the fluorescence sensing platform for probing the high affinity of the short aptamer derived from the wild-type long aptamer-CYN sensing. The biosensor construction involved two steps: first, quenching the fluorescence of fluorescent-labelled truncated aptamer using GO as a quencher and, second, fluorescence recovery in the presence of CYN by competitive binding between the target and GO. One of the truncate aptamers has a 12-fold higher affinity and enhances sensitivity compared to the long aptamer sequence. The limit of detection of the high affinity truncated aptamer is 17pM which is 6-fold lower than the long aptamer (100pM). The sensor specifically detects CYN in the presence of other potential interfering toxins. The performance of the sensor was validated using CYN spiked tap water with very good recovery percentage. A rapid and highly sensitive detection of CYN from water resources has been achieved using this method.

Global scanning of cylindrospermopsin: Critical review and analysis of aquatic occurrence, bioaccumulation, toxicity and health hazards

Cylindrospermopsin (CYN), a cyanotoxin produced by harmful algal blooms, has been reported worldwide; however, there remains limited understanding of its potential risks to surface water quality. In the present study, we critically reviewed available literature regarding the global occurrence, bioaccumulation, and toxicity of CYN in aquatic systems with a particular focus on freshwater. We subsequently developed environmental exposure distributions (EEDs) for CYN in surface waters and performed probabilistic environmental hazard assessments (PEHAs) using guideline values (GVs). PEHAs were performed by geographic region, type of aquatic system, and matrix. CYN occurrence was prevalent in North America, Europe, and Asia/Pacific, with lakes being the most common system. Many global whole water EEDs exceeded guideline values (GV) previously developed for drinking water (e.g., 0.5 μg L−1) and recreational water (e.g., 1 μg L−1). GV exceedances were higher in the Asia/Pacific region, and in rivers and reservoirs. Rivers in the Asia/Pacific region exceeded the lowest drinking water GV 73.2% of the time. However, lack of standardized protocols used for analyses was alarming, which warrants improvement in future studies. In addition, bioaccumulation of CYN has been reported in mollusks, crustaceans, and fish, but such exposure information remains limited. Though several publications have reported aquatic toxicity of CYN, there is limited chronic aquatic toxicity data, especially for higher trophic level organisms. Most aquatic toxicity studies have not employed standardized experimental designs, failed to analytically verify treatment levels, and did not report purity of CYN used for experiments; therefore, existing data are insufficient to derive water quality guidelines. Considering such elevated exceedances of CYN in global surface waters and limited aquatic bioaccumulation and toxicity data, further aquatic monitoring, environmental fate and mechanistic toxicology studies are warranted to robustly assess and manage water quality risks to public health and the environment.

Cylindrospermopsin induces abnormal vascular development through impairing cytoskeleton and promoting vascular endothelial cell apoptosis by the Rho/ROCK signaling pathway

Cylindrospermopsin (CYN) is a widely distributed cyanobacterial toxin in water bodies and is considered to pose growing threats to human and environmental health. Although its potential toxicity has been reported, its effects on the vascular system are poorly understood. In this study, we examined the toxic effects of CYN on vascular development and the possible mechanism of vascular toxicity induced by CYN using zebrafish embryos and human umbilical vein endothelial cells (HUVECs). CYN exposure induced abnormal vascular development and led to an increase in the growth of common cardinal vein (CCV), in which CCV remodeling was delayed as reflected by the larger CCV area and wider ventral diameter. CYN decreased HUVECs viability, inhibited HUVECs migration, promoted HUVECs apoptosis, destroyed cytoskeleton, and increased intracellular ROS levels. Additionally, CYN could promote the expression of Bax, Bcl-2, and MLC-1 and inhibit the expression of ITGB1, Rho, ROCK, and VIM-1. Taken together, CYN may induce cytoskeleton damage and promote vascular endothelial cell apoptosis by the Rho/ROCK signaling pathway, leading to abnormal vascular development. The current results provide potential insight into the mechanism of CYN toxicity in angiocardiopathy and are beneficial for understanding the environmental risks of CYN for aquatic organisms and human health.

Adverse effect of cylindrospermopsin on embryonic development in zebrafish (Danio rerio).

Eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is receiving great interest due to its increasing presence in waterbodies. However, the toxic effects of CYN on zebrafish development are poorly understood, especially the toxicological mechanism, which is still unclear. In this study, we examined the adverse effects of CYN on embryonic development in zebrafish. CYN (2-2000 nM) exposure decreased embryos survival rate, hatching rate, body length and eye size in a concentration-dependent manner and caused abnormalities in embryo morphology, including pericardial edema, spinal curvature, tail deformity, uninflated swim bladder, cardiac and vascular defects. CYN at concentrations of 20 nM or higher significantly increased ROS level and promoted cell apoptosis in zebrafish embryos. To preliminarily elucidate the potential mechanism of zebrafish developmental toxicity caused by CYN, we examined the expression of oxidative stress- and apoptotic-related genes. CYN could promote the expression of oxidative stress-related genes (SOD1, CAT and GPx1) and induce changes in transcriptional levels of apoptotic-related genes (p53, Bax and Bcl-2). Taken together, CYN induced adverse effects on zebrafish embryos development, which may associate with oxidative stress and apoptosis. These outcomes will advance our understanding of CYN toxicity, environmental problems and health hazards caused by climate changes and eutrophication.

Elucidating cylindrospermopsin toxicity via synthetic analogues: An in vitro approach

Cylindrospermopsin (CYN) is an alkaloid biosynthesized by selected cyanobacteria, the cyto- and genotoxic properties of which have been studied extensively by in vitro and in vivo experimental models. Various studies have separately established the role of uracil, guanidine and hydroxyl groups in CYN-induced toxicity. In the present study, we have prepared five synthetic analogues that all possess a uracil group but had variations in the other functionality found in CYN. We compared the in vitro toxicity of these analogues in common carp hepatocytes by assessing oxidative stress markers, DNA fragmentation and apoptosis. All the analogues tested induced generation of reactive oxygen species, lipid peroxidation (LPO) and DNA fragmentation. However, the greatest increase in LPO and increase in caspase-3 activity, an apoptosis marker, was demonstrated by an analogue containing guanidine, hydroxyl and uracil functionalities similar to those found in CYN but lacking the complex tricyclic structure of CYN. We also report a crystal structure of an analogue lacking the hydroxyl group found in CYN which does not show intramolecular H-bonding interactions between the guanidine and the uracil functionalities. The observations made in this work supports the hypothesis that CYN toxicity is a result of an interplay between both of the uracil, hydroxyl and guanidine functional groups.

Effects of cylindrospermopsin on cultured immortalized human airway epithelial cells

Anthropogenic eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is detected in the environment with increasing frequency, driving the scientific effort to assess emerging health risks from CYN-producing blooms. Oral exposure to CYN results primarily in hepatotoxicity. Nevertheless, extrahepatic manifestations of CYN toxicity have been reported. Furthermore, cyanotoxins have been detected in aerosols and dust particles, suggesting potential toxic effects in the respiratory tract. To assess the susceptibility of airway epithelia towards cyanotoxins, monolayers of immortalized human bronchial epithelial cells HBE1 and 16HBE14o- were exposed to a concentration range of 0.1–10 μM CYN. Cytotoxic endpoints were assessed as morphologic alterations, resazurin reduction capacity, esterase activity, neutral red uptake, and by impedimetric real-time cell analysis. Depending on the endpoint assessed, EC50 values ranged between 0.7 and 1.8 μM (HBE1) and 1.6–4.8 μM (16HBE14o-). To evaluate alterations of other cellular events by subcytotoxic concentration of CYN (1 μM), phosphorylation of mitogen-activated protein kinases ERK and p38 was determined. Only a slight increase in p38 phosphorylation was induced by CYN in HBE1 cell line after 48 h, while activities of both ERK1/2 and p38 gradually and significantly increased in 16HBE14o- cells during 8–48 h exposure. This study suggests possible hazards of inhalation CYN exposures, which may severely impact the integrity of airway epithelia and epithelial cell signaling. Further research of CYN-induced toxicity and underlying mechanisms is needed, as well as more data on environmental concentrations of cyanotoxins in aerosols for exposure assessment.

Synthetic analogues of cyanobacterial alkaloid cylindrospermopsin and their toxicological activity

Cylindrospermopsin (CYN) is a naturally occurring alkaloid produced by a variety of cyanobacteria and known to induce oxidative stress-mediated toxicity in eukaryotic cells. Despite extensive research on the mechanism of CYN toxicity, an understanding of the structural features responsible for this toxicity and the mechanism by which it can enter the cell are still not clear. It was established that the presence of both the uracil and guanidine groups is essential in biological activity of CYN whilst not much is known in this regard on the role of tether that separates them and the attached hydroxyl group. Therefore, in the present study we have prepared three synthetic analogues possessing uracil and guanidine groups separated by a variable length tether (4–6 carbons) and containing a hydroxyl function in a position orientation to CYN, together with a tetracyclic analogue of CYN lacking the hydroxyl group at C-7. The toxicity of these compounds was then compared with CYN and guanidinoacetate (GAA; the primary substrate in CYN biosynthesis) in an in vitro model using human neutrophils isolated from healthy subjects. The lowest activity measured by means of reactive oxygen species generation, lipid peroxidation and cell death was observed for GAA and the tetracyclic analogue. The greatest toxicity was found in an analogue with a 6-carbon tether, but all three analogues and CYN caused rapid onset of redox imbalance. These results add to the general understanding of CYN toxicity and preliminary findings suggest that the –OH group at C-7 may be significant for the cellular transport of CYN and/or be involved in its toxic activity inside the cell, a hypothesis which requires further testing.

The interactive effects of microcystin-LR and cylindrospermopsin on the growth rate of the freshwater algae Chlorella vulgaris.

Microcystin-LR (MC-LR) and cylindrospermopsin (CYN) are the most representative cyanobacterial cyanotoxins. They have been simultaneously detected in aquatic systems, but their combined ecotoxicological effects to aquatic organisms, especially microalgae, is unknown. In this study, we examined the effects of these cyanotoxins individually and as a binary mixture on the growth rate of the freshwater algae Chlorella vulgaris. Using the MIXTOX tool, the reference model concentration addition (CA) was selected to evaluate the combined effects of MC-LR and CYN on the growth of the freshwater green algae due to its conservative prediction of mixture effect for putative similar or dissimilar acting chemicals. Deviations from the CA model such as synergism/antagonism, dose-ratio and dose-level dependency were also assessed. In single exposures, our results demonstrated that MC-LR and CYN had different impacts on the growth rates of C. vulgaris at the highest tested concentrations, being CYN the most toxic. In the mixture exposure trial, MC-LR and CYN showed a synergistic deviation from the conceptual model CA as the best descriptive model. MC-LR individually was not toxic even at high concentrations (37mgL(-1)); however, the presence of MC-LR at much lower concentrations (0.4-16.7mgL(-1)) increased the CYN toxicity. From these results, the combined exposure of MC-LR and CYN should be considered for risk assessment of mixtures as the toxicity may be underestimated when looking only at the single cyanotoxins and not their combination. This study also represents an important step to understand the interactions among MC-LR and CYN detected previously in aquatic systems.

Antioxidative stress responses in the floating macrophyte Lemna minor L. with cylindrospermopsin exposure

Cylindrospermopsin toxicity and oxidative stress have been examined in aquatic animals, however, only a few studies with aquatic plants have been conducted focusing on the potential for bioaccumulation of cylindrospermopsin. The oxidative stress effects caused by cylindrospermopsin on macrophytes have not yet been specifically studied. The oxidative stress response of Lemna minor L. with exposure to cylindrospermopsin, was therefore tested in this study. The hydrogen peroxide concentration together with the activities of the antioxidant enzymes (catalase, peroxidase, glutathione reductase and glutathione S-transferase) were determined after 24h (hours) of exposure to varying concentrations (0.025, 0.25, 2.5 and 25μg/L) of cylindrospermopsin. Responses with longer exposure periods (48, 96, 168h) were tested only with exposure to 2.5 and 25μg/L cylindrospermopsin. Additionally, the content of the carotenoids was determined as a possible non-enzymatic antioxidant defence mechanism against cylindrospermopsin. The levels of hydrogen peroxide increased after 24h even at the lowest cylindrospermopsin exposure concentrations. Catalase showed the most representative antioxidant response observed after 24h and maintained its activity throughout the experiment. Catalase activity corresponded with the contents of hydrogen peroxide at 2.5 and 25μg/L cylindrospermopsin. The data suggest that glutathione S-transferase, glutathione reductase and the carotenoid content act together with catalase but are more sensitive to higher concentrations of cylindrospermopsin and after a longer exposure period (168h). The results indicate that cylindrospermopsin promotes oxidative stress in L. minor at concentrations of 2.5 and 25μg/L. However, L. minor has sufficient defence mechanisms in place against this cyanobacterial toxin. Even though L. minor exhibits the potential to managing and control cylindrospermopsin contamination in aquatic systems, further studies in tolerance limits to cylindrospermopsin, uptake and experiments with prolonged exposure periods of more than 7 days are required.

Degradation of cyanotoxin cylindrospermopsin by TiO2-assisted ozonation in water

The stable tricyclic structure of the cylindrospermopsin (CYN), a cynotoxin, has presented several challenges to water treatment facilities, as conventional treatment methods have a limited ability to remove it from water. This study examines the effectiveness of titanium dioxide (TiO2) in catalytic ozonation for degrading CYN. The chemical kinetics of the reactions of ozone (O3) and hydroxyl radicals (OH•) with CYN were determined. The results reveal that TiO2 significantly increases the rate of degradation of CYN by increasing the rate of production of hydroxyl radicals (OH•) by initiating the decomposition of O3 on the surface of the catalyst. At a pH of 7 with 1.0 mg L−1 O3 and 500 mg L−1 TiO2; the pseudo–first-order ozone decomposition rate constant (kD) increased from 3.04 × 10−3 to 16.53 × 10−3 s−1 and the ratio of OH• to O3 concentrations (Rct) increased from 1.87 × 10−8 to 126.4 × 10−8. The calculated second-order rate constant (koverall) of the reaction of CYN with O3 and OH• was 3.22 M−1s−1 without TiO2. However, the greatest improvement in koverall in this study was observed using 500 mg TiO2 L−1, which increased koverall by a factor of five. TiO2-catalyzed ozonation is an efficient method of oxidation that reduces the toxic activity of CYN. The results of a Microtox test concerning the toxic activity of CYN during oxidation reveal that catalytic ozonation may either increase or reduce the toxicity of CYN toward test samples. The toxic effects of CYN on the samples are greatly influenced by the TiO2 dosage and reaction time, possibly yielding by-products that may change the mutagenic properties of CYN. Three water samples from a eutrophic lake in Taiwan were examined to evaluate the effect of dissolved organic carbon (DOC) and alkalinity on the oxidation of CYN. DOC had the greatest effect on the oxidation of CYN in the ozonation of eutrophic water. Overall, the degree of CYN oxidation depended on the rate constant of the reaction with ozone and the consumption of ozone by the natural water matrix.

Modulation of chromatin remodelling induced by the freshwater cyanotoxin cylindrospermopsin in human intestinal caco-2 cells.

Cylindrospermopsin (CYN) is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 µM for 24hrs) using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes), and DNA recombination and repair (with up-regulation of aptx and pms2 genes). Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2) involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5) and dimethylated histone H3 (Lys4), two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN.

Toxicity of cylindrospermopsin in human lymphocytes: Proliferation, viability and cell cycle studies

The global expansion of cylindrospermopsin (CYN) producing cyanobacteria in surface freshwater increases the risk of human exposure and poisoning. Following ingestion, CYN is transported with blood in general circulation to the liver and kidneys, and can potentially interact with immune system cells. In the present study, we investigated whether CYN (0.01–1.0μgml−1) can alter the function of human peripheral blood lymphocytes isolated from healthy donors. It was found that CYN demonstrates significant antiproliferative activity in lymphocytes during different phases of their activation. The most remarkable effects (decrease by>90%) were observed in lymphocytes exposed to 1μgml−1 CYN at the beginning of activation. Further analyses revealed a cell-cycle arrest at G0/G1 and prolonged S phase in lymphocytes undergoing activation and significant apoptosis inducement in activated cells. Reduced abilities to fight pathogenic microorganisms or malignant cells should be taken into consideration in CYN exposure and risk assessments.

Immunohistochemical Approach to Study Cylindrospermopsin Distribution in Tilapia (Oreochromis niloticus) under Different Exposure Conditions

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.