Nodularin Research Articles

Modulation of nodularin toxicity in shrimp Litopenaeus vannamei (BOONE, 1931) fed with dietary açai (Euterpe oleracea) inclusion

This study evaluated the effect of dietary inclusion of lyophilized açaí Euterpe oleracea (LEO) on redox status of shrimp Litopenaeus vannamei (initial weight 1.5 ± 0.39 g) upon exposure to cyanotoxin nodularin (NOD) in bioflocs system. Three hundred juvenile shrimps were randomly divided into two groups and fed twice a day with two diets: one containing 0.00 (control diet) and the other 10.0% LEO (w/w) for 30-days. After the feeding period, both shrimp groups were submitted to three treatments (14 L; 7 shrimp/tank) with different concentrations of cyanotoxin NOD (0.00; 0.25; and 1.00 μg/L) dissolved in water with 96 h of exposure. Then, the shrimps were sampled (n = 15/treatment) for the determination of reduced glutathione (GSH), the activity of glutathione-S-transferase (GST), sulfhydryl groups associated to proteins (P-SH), and lipid peroxidation (TBARS) in the hepatopancreas, gills and muscle. The NOD accumulation was measured in the muscle. The results revealed that dietary LEO significantly increased GSH levels in the hepatopancreas and gills of the shrimps exposed to NOD. Toxin exposure did not modify GST activity in all organs. Muscle TBARS levels were lower in the shrimp fed with the LEO diet and exposed to NOD. The NOD toxin did not accumulate in the muscle but notably was detected in the control groups fed or not with dietary LEO. Açaí was able to induce the antioxidant system of L. vannamei, as well as lowered the oxidative damage in shrimps exposed to NOD, suggesting its use as a chemoprotectant against cyanotoxins.

Synthesis of metal-organic framework-5@chitosan material for the analysis of microcystins and nodularin based on ultra-performance liquid chromatography-tandem mass spectrometry

Microcystins (MCs) and nodularin (NOD) are tumor promoters produced by cyanobacteria and present in surface water. In this work, a novel mesoporous metal-organic framework-5@chitosan (MOF-5@CS) material was synthesized and applied for the enrichment of MCs and NOD in water and fish samples. The mesoporous MOF-5@CS material was firstly synthesized via a one-step hydrothermal method, and the chitosan was combined with MOF-5 via chemical bonding assembly. As a new adsorbent, the as-synthesized material was found having a large specific surface area and good thermal stability. Under the optimized conditions, MCs and NOD were enriched by the MOF-5@CS material and detected by ultra-performance liquid chromatography-tandem mass spectrometry. The limit of detection of the new method for MCs and NOD were in the range of 0.0018-0.077 ng/mL. The value of relative standard deviation for repeatability were 2.69-6.30%, and the recovery of the analytes ranged from 84.36% to 118.51%. Compared with other reported method for MCs and NOD detection in complex matrices, better adsorption performance for MCs and NOD were obtained by our new method, and the sensitivity of MCs-RR and NOD were improved nearly 20 times and 30 times, respectively.

Occurrence, quantification, and adsorptive removal of nodularin in seawater, wastewater and river water

The presence of potent hepatotoxic cyanotoxins such as nodularin (NOD) in drinking water, groundwater, surface water, seawater and recreational waters presents a major risks to human and environmental health. Human exposure to cyanotoxins could lead to various health effects such as liver damage, jaundice, neurotoxicity and gastroenteritis. Therefore, it is critical to investigate their occurrence in environmental matrices. This study reports the use of tyre-based activated carbon (WTAC) as an adsorbent for preconcentration and removal of nodularin from environmental matrices prior to high performance liquid chromatographic analysis. The preconcentration and adsorption experiments were carried out in presence of other environmental components to consider the external effect on WTAC adsorption of nodularin. Under optimum conditions, the linear dynamic range was 0.05–70 μg L− 1 with a correlation coefficient of 0.9991. The LOD and LOQ (n = 10) in the absence and in the presence of humic acids were 0.012–0.025 μg L− 1 and 0.040–0.083, n = 10), respectively. The repeatability (n = 10) and reproducibility (n = 5) of the method expressed as relative standard deviation (%RSD) were 3.7 and 5.1%, respectively. The maximum adsorption capacity of WTAC was 345 μg g −1. Furthermore, the results demonstrated that the presence of humic acid has an effect on the nodularin adsorption to WTAC. However, high concentrations other coexisting ions such a Cl−, NO3−, PO43−, HCO3−, SO42− had no significant the effect on the adsorption process. The proposed technique was then used for a preconcentration and elimination of NOD trace levels in different water matrices. The results showed that the WTAC was an effective adsorbent for the preconcentration and removal of NOD from the complex matrices.

Purification and activity of the first recombinant enzyme for biodegrading hepatotoxin by Sphingopyxis sp. USTB-05

Cyanobacterial hepatotoxins which are produced and released by bloom-forming cyanobacteria may threaten the safety of drinking water for human beings and animals. Microcystinase (MlrA) catalyzes the first and most important step for biodegrading hepatotoxic microcystins (MCs) and nodularin (NOD), which hydrolyzes the cyclic hepatotoxin into the linear hepatotoxin as the first product. Here the recombinant MlrA of Sphingopyxis sp. USTB-05 for biodegrading hepatotoxins was firstly purified and its activity was also investigated. The purification process of MlrA was comprised of four steps, affinity purification of glutathione S-transferase (GST)-tagged MlrA, thrombin cleavage of GST tag, GST tag removal and thrombin removal with the benzamidine sepharose. The purified recombinant MlrA was found to have a strong ability to catalyze hepatotoxins, and initial microcystin-LR (MC-LR) of 19.9 mg/L and NOD of 25.2 mg/L were removed within 10 min and 12 h at the protein concentration of 60 mg/L, respectively. The results indicated that the enzyme MlrA with a high activity could be purified and obtained with the biotechnology process of expression and purification described here, which is very important in further studies on the enzyme structure and the enzymatic mechanism for biodegrading hepatotoxins.

Nodularin induced oxidative stress contributes to developmental toxicity in zebrafish embryos.

Nodularin (NOD) is a kind of cyanobacterial toxins. It is of concern due to elicit severe genotoxicity in humans and animals. The comprehensive evaluation of NOD-induced adverse effects in living organisms is urgently needed. This study is aimed to report the developmental toxicity and molecular mechanism using zebrafish embryos exposed to NOD. The embryonic toxicity induced by NOD is demonstrated by inhibition of embryo hatching, increase in mortality rate, abnormal heart rate, embryonic malformation as well as defects in angiogenesis and common cardinal vein remodeling. NOD triggered a decreased rate of angiogenesis through inhibiting endothelial cells migration. NOD induced embryonic cell apoptosis and DNA damage, which can be alleviated by antioxidant N-acetyl-L-cysteine. NOD significantly caused oxidative damage as indicated by changes in reactive oxygen species, superoxide dismutase, catalase, glutathione and malondialdehyde. NOD also altered the expression of vascular development-genes (DLL4, CDH5, VEGFA, VEGFC) and apoptosis-related genes (BAX, BCL-2, P53, CASPASE 3). Taken together, NOD induced adverse effect on zebrafish embryos development, which may be associated with oxidative stress and apoptosis through the activation of P53-BAX/BCL-2-CASPASE 3-mediated pathway.

Phosphate and nitrate supplementations to evaluate the effect on cell biomass, intra and extracellular nodularin and nodulopeptin 901 produced by the cyanobacterium Nodularia spumigena KAC 66

Blooms of the cyanobacterium Nodularia spumigena occur regularly in the Baltic Sea typically producing a wide range of bioactive peptides including the hepatotoxin nodularin (NOD), spumigins, anabaenopeptins and nodulopeptins (molecular weights: 917, 901 and 899 Da). This study reports the production of intracellular and extracellular NOD and nodulopeptin 901 (the major secondary metabolites), at various nitrate and phosphorus concentrations produced by N. spumigena KAC 66 which had been originally isolated from the Baltic Sea. The growth was observed by cell biomass and intracellular and extracellular peptides monitored by high-performance liquid chromatography with photodiode array and mass spectrometry (HPLC-PDA-MS). In the present work, it was found that high concentrations of nitrate and phosphorus have a considerable effect on biomass and toxin levels of N. spumigena. In common with many studies, the maximum amount of NOD was retained within the cells during 5 weeks of growth. In contrast, as much as ~ 40% of nodulopeptin 901 was excreted into the medium throughout the duration of experiments. At 6.5 and 3.5 mg L−1 nitrate, the maximum concentrations of peptide per unit biomass was 1.78 ng NOD (in week 4) and 1.42 ng nodulopeptin 901 μg−1 (in week 3) were detected. However, the high concentrations of both peptides were produced in the absence of nitrate. The phosphate experiment indicated growth, and peptide production was dependent on availability of phosphorus. At 0 mg L−1 of phosphate, an increased amount of intracellular (502.4 ng μg−1 biomass) nodulopeptin 901 was recorded. This report evaluates the effect of nutrients on the production of biomass and toxins, which may predict the formation and control of blooms of N. spumigena in the Baltic Sea. It also provides information to improve the growth conditions to produce high biomass and toxins under suitable conditions, which may be helpful in the research. The results from the current study will also be helpful to predict about possible blooms of N. spumigena in the Baltic Sea with reference to increase or decrease in nitrate and phosphate concentrations.

Measurement of Microcystin and Nodularin Activity in Human Urine by Immunocapture-Protein Phosphatase 2A Assay

Microcystins (MC) and nodularin (NOD) are toxins released by cyanobacteria during harmful algal blooms. They are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A) and cause a variety of adverse symptoms in humans and animals if ingested. More than 250 chemically diverse congeners of MCs have been identified, but certified reference materials are only available for a few. A diagnostic test that does not require each reference material for detection is necessary to identify human exposures. To address this need, our lab has developed a method that uses an antibody to specifically isolate MCs and NOD from urine prior to detection via a commercially available PP2A kit. This assay quantitates the summed inhibitory activity of nearly all MCs and NOD on PP2A relative to a common MC congener, microcystin-LR (MC-LR). The quantitation range for MC-LR using this method is from 0.050–0.500 ng/mL. No background responses were detected in a convenience set of 50 individual urines. Interday and intraday % accuracies ranged from 94%–118% and relative standard deviations were 15% or less, meeting FDA guidelines for receptor binding assays. The assay detected low levels of MCs in urines from three individuals living in close proximity to harmful algal blooms (HABs) in Florida.

Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin

A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the simultaneous detection of nine microcystins (MCs) and nodularin (NOD) using MC-LR-keyhole limpet hemocyanin (KLH) for New Zealand white rabbit immunization to produce antibodies. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared and heterogeneous coating strategy was found to significantly improve the sensitivity of detection, as evident from the appropriate structure. Comparison of the half-maximum inhibitory concentration (IC50) with MC-LR and MC-LR-BSA coating techniques (0.29 ng/mL) revealed the superior performance of 0.054 ng/mL for NOD-BSA coating. NOD-BSA was selected as the coating antigen, because it showed ultrahigh sensitivity for the detection of MC-LR with a limit of detection (LOD) of 0.0016 ng/mL, which was below the maximum residue level (MRL) of 1 ng/mL. In addition, high reproducibility, good stability, and acceptable spiked sample detection, as validated by liquid chromatography tandem mass spectrometry (LC-MS/MS), indicated the possible application of this method for the analysis of MCs and NOD in water sample.

Effect of the immobilized microcystin-LR-degrading enzyme MlrA on nodularin degradation and its immunotoxicity study

In freshwater ecosystems with frequent cyanobacterial blooms, the cyanobacteria toxin pollution is becoming increasingly serious. Nodularin (NOD), which has strong biological toxicity, has emerged as a new pollutant and affects the normal growth, development and reproduction of aquatic organisms. However, little information is available regarding this toxin. In this study, a graphene oxide material modified by L-cysteine was synthesized and used to immobilize microcystin-LR (MC-LR)-degrading enzyme (MlrA) to form an immobilized enzyme nanocomposite, CysGO-MlrA. Free-MlrA was used as a control. The efficiency of NOD removal by CysGO-MlrA was investigated. Additionally, the effects of CysGO-MlrA and the NOD degradation product on zebrafish lymphocytes were detected to determine the biological toxicity of these two substances. The results showed the following: (1) There was no significant difference in the degradation efficiency of NOD between CysGO-MlrA and free-MlrA; the degradation rate of both was greater than 80% at 1 h (2) The degradation efficiency of the enzyme could retain greater than 81% of the initial degradation efficiency after the CysGO-MlrA had been reused 7 times. (3) CysGO-MlrA retained greater than 50% of its activity on the 8th day when preserved at 0 °C, while free-MlrA lost 50% of its activity on the 4th day. (4) CysGO-MlrA and the degradation product of NOD showed no obvious cytotoxicity to zebrafish lymphocytes. Therefore, CysGO-MlrA might be used as an efficient and ecologically safe degradation material for NOD.

Cloning and Expression of Genes for Biodegrading Nodularin by Sphingopyxis sp. USTB-05.

Biodegradation is efficient for removing cyanobacterial toxins, such as microcystins (MCs) and nodularin (NOD). However, not all the microbial strains with the microcystin-biodegrading enzymes MlrA and MlrC could biodegrade NOD. Studies on genes and enzymes for biodegrading NOD can reveal the function and the biodegradation pathway of NOD. Based on successful cloning and expression of the USTB-05-A and USTB-05-C genes from Sphingopyxis sp. USTB-05, which are responsible for the biodegradation of MCs, the pathway for biodegrading NOD by these two enzymes was investigated in this study. The findings showed that the enzyme USTB-05-A converted cyclic NOD (m/z 825.4516) into its linear type as the first product by hydrolyzing the arginine and Adda peptide bond, and that USTB-05-C cut off the Adda and glutamic acid peptide bond of linearized NOD (m/z 843.4616) and produced dimeric Adda (m/z 663.4377) as the second product. Further, based on the homology modeling of enzyme USTB-05-A, site-directed mutants of USTB-05-A were constructed and seven crucial sites for enzyme USTB-05-A activity were found. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A in the first step was proposed: glutamic acid 172 and histidine 205 activate a water molecule facilitating a nucleophilic attack on the arginine and Adda peptide bond of NOD; tryptophan 176 and tryptophan 201 contact the carboxylate side chain of glutamic acid 172 and accelerate the reaction rates; and histidine 260 and asparagine 264 function as an oxyanion hole to stabilize the transition states.

Development of a validated direct injection-liquid chromatographic tandem mass spectrometric method under negative electrospray ionization for quantitation of nine microcystins and nodularin-R in lake water

Microcystins (MCs) are cyclic heptapeptide toxins produced by various cyanobacterial genera that are toxic to both animals and humans. In this study, a novel strategy was proposed for the quantitation of nine MCs and Nodularin-R (NOD) in lake water using UHPLC-MS/MS under negative ionization mode, in which only centrifugation was employed during sample preparation. As a result, limits of quantification (LOQ) ranging from 0.05 to 0.1 μg/L for all studied compounds were obtained in water samples, which were lower than the results obtained using positive ionization mode. Additionally, validation was performed by spiking three different levels of MCs at 0.05 or 0.1, 0.5, 1.0 μg/L (n = 6). Recoveries ranged from 88.6% to 101.8%, and intraday and interday variability were lower than 12% and 14%, respectively, for all targeted compounds. Furthermore, the proposed method was applied to investigate microcystins contamination in fifty lake water samples collected in different regions in China. As a result, MC-LR, MC-RR, MC-YR, MC-WR, MC-LW, MC-LA, MC-LY, and MC-HilR were detected in lake water samples at trace level ranging from 0.06 to 0.37 μg/L. The obtained results indicated that it was necessary to monitor the presence of MCs in lake water, especially during regular cyanobacterial blooms during warmer months.

Monitoring of cyanotoxins in water from hypersaline microalgae colonies by ultra high performance liquid chromatography with diode array and tandem mass spectrometry detection following salting-out liquid-liquid extraction

In this study two different analytical approaches have been developed to determine the presence of several cyanotoxins in saline water samples from a continental salt marsh. A salting-out assisted liquid-liquid extraction (SALLE) has been used in combination with ultra-high performance liquid chromatography-tandem mass spectrometry and UV-diode array detection (UHPLC-MS/MS and UHPLC-DAD). The target analytes are eight microcystins named MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, MC-LF and nodularin (NOD), covering a wide range of polarities. The separation was achieved using a Zorbax Eclipse Plus RRHD C18 column (50 × 2.1 mm, 1.8 μm) in less than 7.5 and 5.5 min for UV and MS/MS detection, respectively. The mobile phase used consisted of water (solvent A) and acetonitrile (MeCN) (solvent B), both containing 0.01% of formic acid for DAD and 0.4% of formic acid for MS/MS detection, at a flow rate of 0.4 mL min−1. The temperature of the column was set at 25 °C and 20 μL of sample were injected. The main parameters affecting the SALLE procedure were studied and the following optimum values were obtained: neutral pH, 2 mL of acetonitrile as extraction solvent and 1.2 g of ammonium sulfate as salting-out agent for 4 mL of water sample. The validation protocols for both methods were accomplished with real water samples obtaining LODs ranging from 1.0 to 3.4 μg L−1 and 0.02 to 0.11 μg L−1 for DAD and MS/MS respectively. Although the SALLE-UHPLC-DAD methodology is easier and cheaper than UHPLC-MS/MS significantly better detection limits were achieved with tandem mass spectrometry as well as allowing for unambiguous identification. Extraction recoveries were higher than 77.0% (except for MC-RR and NOD which were 53.2% and 54.3, respectively) with satisfactory inter-day and intra-day precisions (RSD below 13.3%).

Aptamer-Based Fluorescent Sensor Array for Multiplexed Detection of Cyanotoxins on a Smartphone.

Developing easy-to-use and miniaturized detectors is essential for in-field monitoring of environmentally hazardous substances, such as the cyanotoxins. We demonstrated a differential fluorescent sensor array made of aptamers and single-stranded DNA (ssDNA) dyes for multiplexed detection and discrimination of four common cyanotoxins with an ordinary smartphone within 5 min of reaction. The assay reagents were preloaded and dried in a microfluidic chip with a long shelf life over 60 days. Upon the addition of analyte solutions, competitive binding of cyanotoxin to the specific aptamer-dye conjugate occurred. A zone-specific and concentration-dependent reduction in the green fluorescence was observed as a result of the aptamer conformation change. The aptasensors are fully optimized by quantification of their dissociation constants, tuning the stoichiometric ratios of reaction mixtures, and implementation of an internal intensity correction step. The fluorescent sensor array allowed for accurate identification and measurement of four important cyanotoxins, including anatoxin-a (ATX), cylindrospermopsin (CYN), nodularin (NOD), and microcystin-LR (MC-LR), in parallel, with the limit of detection (LOD) down to a few nanomolar (<3 nM), which is close to the World Health Organization's guideline for the maximum concentration allowed in drinking water. The smartphone-based sensor platform also showed remarkable chemical specificity against potential interfering agents in water. The performance of the system was tested and validated with real lake water samples that were contaminated with trace levels of individual cyanotoxins as well as binary, ternary, and quaternary mixtures. Finally, a smartphone app interface has been developed for rapid on-site data processing and result display.

Detected cyanotoxins by UHPLC MS/MS technique in tropical reservoirs of northeastern Colombia

This study focused on the detection and quantification of eight cyanotoxins in water samples in three reservoirs located in the eastern department of Antioquia, Colombia. The reservoirs are a source of water supply and hydroelectricity, and also generate economic activities in fishing and recreation. Between May 2015 and October 2016, 8 samplings were carried out at times of high temperatures, which ranged from 20 to 29 °C. This period was selected because of a significant or strong El Niño phenomenon, according to the World Meteorological Organization. For the study, 270 integrated samples were taken from the photic zone (PZ) and the surface of the reservoirs, at each of the three sampling points. The samples were analyzed by the analytical technique of ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC MS/MS). The quantification performed for six microcystins (MCs), a nodularin (NOD) and a cylindrospermopsin (CYN), showed positive results well above 1 μg L−1. In the water of the Abreo Malpaso and Peñol reservoirs, microcystin-LR (MC-LR), microcystin-YR (MC-YR) and [D-Asp3,(E)-Dhb7]- microcystin-RR toxins were detected at levels of considerable concentration, especially between May and September 2015, when there was no rainfall in this region. In the Playas reservoir, positive results for [D-Asp3,(E)-Dhb7]-MC-RR were detected from May to November 2015, with the highest concentrations being reached in dry season. The temperatures reached and the changes in climatic conditions witnessed during the monitoring period of this study were important factors in the production of cyanotoxins. This was evidenced in this work by the high concentrations of detected cyanotoxins and their absence in periods of rain, as happened from the second quarter of 2016 until the end of the study in October. This is the first study of the detection and quantification of cyanotoxins in tropical reservoirs of northeastern Colombia using the UHPLC MS/MS analytical technique, which allowed the toxins to be unequivocally detected and confirmed. A method was developed and validated, proving to be sensitive, reproducible and accurate. For each of the toxins (microcystin-LR (MC-LR), microcystin-RR (MC-RR), microcystin-YR (MC-YR), [D-Asp3,(E)-Dhb7]- microcystin-RR, microcystin-LW (MC-LW), microcystin-LF (MC-LF), nodularin (NOD) and cylindrospermopsina (CYN)) the correlation coefficients (R2) were in a range between 0.9907 and 0.9999. Verification of the accuracy of the method was performed through a calibration curve in solvent. The recovery percentages of the accuracy and precision tests of the method for low level, medium level and high level were in a range between 64% and 115% for all the cyanotoxins. The validation of the cyanotoxin method shows that it is possible to detect them individually in natural water with a quantification limit (LOQ) of approximately 0.05 μg L−1.

Study of cyanotoxin degradation and evaluation of their transformation products in surface waters by LC-QTOF MS

In the present work, the degradation of three cyanotoxins from the hepatotoxins group was investigated under laboratory-controlled experiments in water samples. Surface waters spiked with microcystin-LR (MC-LR), nodularin (NOD) and cylindrospermopsin (CYN) were subjected to hydrolysis, chlorination and photo-degradation, under both sunlight (SL) and ultraviolet (UV) radiation. A total of 12 transformation products (TPs) were detected and tentatively identified by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS). These comprised: 6 chlorination TPs (3 from CYN and 3 from MC-LR, 2 isomers); 4 UV TPs (all from CYN); and 2 sunlight TPs (one isomer from MC-LR and another from NOD). No TPs were observed under hydrolysis conditions. The chemical structures for all TPs were tentatively proposed based on the accurate-mass QTOF MS full-spectra. Analysis of real-world samples collected from the Peñol reservoir (Antioquia, Colombia) revealed the presence of MC-LR and CYN as well as a sunlight TP identified in the laboratory experiments. Data presented in this article will assist further research on TPs potentially formed in future tertiary degradation processes applied for the removal of organic micro-pollutants in water; as well as improving available knowledge on the toxic implications of cyanobacterial toxins TPs in surface waters.

Determination of cylindrospermopsin, nodularin and microcystins in freshwater fish by dispersive solid phase extraction-liquid chromatography-tandem mass spectrometry

A method was developed for the determination of cylindrospermopsin (CYN), nodularin (NOD), microcystin-RR (MC-RR), microcystin-YR (MC-YR) and microcystin-LR (MC-LR) in freshwater fish by dispersive solid phase extraction-liquid chromatography-tandem mass spectrometry (DSPE-LC-MS/MS). The analytes were extracted from fish tissues with acetonitrile-water-formic acid (89:10:1, v/v/v), and purified by DSPE using C18 as the adsorbent. The separation of analytes was performed on an Agilent ZORBAX Eclipse XDB C18 column with the gradient elution of acetonitrile and water as mobile phases. Qualitative analysis was performed using the multiple reaction monitoring (MRM) mode. The analytes were quantified by matrix-matched external standard curves. The chromatographic and MS parameters were optimized. Major factors affecting the extraction and cleanup efficiencies including the type of extraction solvent and cleanup sorbent were investigated. The linear correlation coefficients (R2) of the five target compounds were no less than 0.9954. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the five target compounds were 5-10 μg/kg and 15-40 μg/kg, respectively. The spiked recoveries of the five target compounds ranged from 62.3% to 101.2%. The developed method is simple, rapid, accurate, sensitive, and is suitable for the determination of cylindrospermopsin, nodularin and microcystins in freshwater fish.

Genotoxic effects of the cyanobacterial pentapeptide nodularin in HepG2 cells

The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1 μg/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.

Occurrence and diversity of cyanotoxins in Greek lakes

Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm3MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754 ng mg−1 dw, followed by MC-LR (458 ng mg−1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria).

Remediation of Nodularin-R via Electrochemical Removal Using Nanostructured PdO-TiO2@Carbon Anodes

Nodularin is a cyanotoxin commonly produced by cyanobacteria in algal blooms. Among Nodularin variants, the most common is Nodularin-R (NOD-R). This toxin is widely detected in water sources worldw...

Biodegradation of nodularin and other nonribosomal peptides by the Baltic bacteria

Microcystins (MCs) and nodularin (NOD), the hepatotoxic nonribosomal peptides (NRPs) produced by cyanobacteria, are considered as natural pollutants of many aquatic ecosystems. Removal of the toxins proceeds mainly through biodegradation and/or photolysis. In this study, microbial degradation of NOD and other NRPs produced by Nodularia spumigena was investigated. Both, natural bacterial consortia and individual strains from the Southern Baltic Sea were used. Bacterial samples were also screened for the presence of mlrA-D genes, previously proved to be involved in the enzymatic breakdown of hepatotoxic cyanopeptides. The Baltic sediments, with natural bacterial consortia, showed NOD-degrading activity throughout the whole year. These samples were also active against other NRPs: anabaenopeptins and spumigins. The time required for NOD biodegradation ranged from 3 to 41 days and depended on the NOD content in the collected sediments. NOD-degrading activity was also revealed in the presence of ten bacterial strains isolated from sediments and three strains isolated from filaments of N. spumigena CCNP1401. The mlrA-D genes were not found in sediments and only individual genes from the entire mlr cluster were detected in five isolated bacteria. The results suggest that in the case of Baltic bacteria other genes are involved in the biosynthesis of NOD-degrading enzymes.