cyanoginosin-lr and microcystin

Microcystins are a family of more than 50 structurally similar hepatotoxins produced by species of freshwater cyanobacteria, primarily Microcystis aeruginosa. They are monocyclic heptapeptides, characterised by some invariant amino acids, including one of unusual structure which is essential for expression of toxicity. Microcystins are chemically stable, but suffer biodegradation in reservoir waters. The most common member of the family, microcystin-LR (L and R identifying the 2 variable amino acids, in this case leucine and arginine respectively) has an LD50 in mice and rats of 36-122 microg/kg by various routes, including aerosol inhalation. Although human illnesses attributed to microcystins include gastroenteritis and allergic/irritation reactions, the primary target of the toxin is the liver, where disruption of the cytoskeleton, consequent on inhibition of protein phosphatases 1 and 2A, causes massive hepatic haemorrhage. Microcystins are tight-binding inhibitors of these protein phosphatases, with inhibition constants in the nanomolar range or lower. Uptake of microcystins into the liver occurs via a carrier-mediated transport system, and several inhibitors of uptake can antagonise the toxic effects of microcystins. The most effective of these is the antibiotic rifampin (a drug approved for clinical use), which protects mice and rats against microcystin-induced lethality when given prophylactically and, in some cases, therapeutically.

Topics: Animals; Bacterial Toxins; Chemical and Drug Induced Liver Injury; Cyanobacteria; Enzyme Inhibitors; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases

Microcystin-LR (MC-LR) is a potent hepatotoxin that can cause liver inflammation and injury. However, the mode of action of related inflammatory factors is not fully understood. PfHMGB1 is an inflammatory factor induced at the mRNA level in the liver of juvenile yellow catfish (Pelteobagrus fulvidraco) that were intraperitoneally injected with 50 μg/kg MC-LR. The PfHMGB1 mRNA level was highest in the liver and muscle among 11 tissues examined. The full-length cDNA sequence of PfHMGB1 was cloned and overexpressed in E. coli, and the purified protein rPfHMGB1 demonstrated DNA binding affinity. Endotoxin-free rPfHMGB1 (6-150 μg/mL) also showed dose-dependent hepatotoxicity and induced inflammatory gene expression of primary hepatocytes. PfHMGB1 antibody (anti-PfHMGB1) in vitro reduced MC-LR (30 and 50 μmol/L)-induced hepatotoxicity, suggesting PfHMGB1 is important in the toxic effects of MC-LR. In vivo study showed that MC-LR upregulated PfHMGB1 protein in the liver. The anti-PfHMGB1 blocked its counterpart and reduced ALT/AST activities after MC-LR exposure. Anti-PfHMGB1 partly neutralized MC-LR-induced hepatocyte disorganization, nucleus shrinkage, mitochondria, and rough endoplasmic reticula destruction. These findings suggest that PfHMGB1 promotes MC-LR-induced liver damage in the yellow catfish. HMGB1 may help protect catfish against widespread microcystin pollution.

Topics: Animals; Catfishes; Chemical and Drug Induced Liver Injury, Chronic; DNA, Complementary; Escherichia coli; Gene Expression; Hepatocytes; Liver; Liver Diseases; Marine Toxins; Microcystins; Proteins; RNA, Messenger

To investigate whether microcystin-LR (MC-LR) influences children's cognitive function and memory ability, we measured serum MC-LR and whole blood lead levels in 697 primary students, and collected their academic and neurobehavioral test scores. The median of serum MC-LR levels was 0.80 µg/L (the value below the limit of detection to 1.67 µg/L). The shapes of the associations of serum MC-LR levels (cut-point: 0.95 µg/L) with scores on academic achievements, digit symbol substitution test and long-term memory test were parabolic curves. Logistic regression analysis showed that MC-LR at concentrations of 0.80-0.95 µg/L was associated with the increased probability of higher achievements on academic achievements [odds ratio (OR) = 2.20, 95% confidence interval (CI): 1.28-3.79], and also with scores on digit symbol substitution test (OR = 1.73, 95% CI: 1.05-2.86), overall memory quotient (OR = 2.27, 95% CI: 1.21-4.26), long-term memory (OR = 1.85, 95% CI: 1.01-3.38) and short-term memory (OR = 2.13, 95% CI: 1.14-3.98) after adjustment for confounding factors. Antagonism of MC-LR and lead on long-term memory was observed (synergism index = 0.15, 95% CI: 0.03-0.74). In conclusion, serum MC-LR at concentrations of 0.80-0.95 µg/L was positively associated with higher scores on cognitive and neurobehavioral tests, and antagonism between MC-LR at concentrations of 0.80-1.67 µg/L and lead exposure was obviously observed on long-term memory in children. Concerning that MC-LR is a neurotoxin at high doses, our observation is interesting and need further investigation.

Topics: Child; China; Cognition; Cross-Sectional Studies; Environmental Exposure; Humans; Lead; Marine Toxins; Memory; Microcystins; Schools; Water Pollutants, Chemical

Freshwater harmful algal blooms (HABs) are a major environmental health problem worldwide. HABs are caused by a predominance of cyanobacteria, some of which produce potent toxins. The most ubiquitous cyanotoxin is microcystin (MC) and the congener MC-LR is the most studied due to its toxicity. Short-term exposure to toxins can cause gut microbiome disturbances, but this has not been well described with MC-LR exposure. This study investigated the gut microbial communities of mice from a prior study, which identified significant liver toxicity from ingestion of MC-LR daily for 8 days. CD-1 mice were divided into three dosage groups: control, low exposure (sub-lethal MC-LR concentration), and high exposure (near-lethal MC-LR concentration). Fecal samples were analyzed using 16S rRNA sequencing. Results revealed that at population level, there were no significant shifts in bacterial diversity or the microbial community structure over the exposure period. However, there were significant differences between male and female mice. Predictive functional gene analysis indicated that several metabolic pathways were significantly different in the high dose group before exposure and following 7 doses of MC-LR, as well as between the control and high dose groups on Day 8. Significant differentially abundant taxa were also identified contributing to these pathways. Several pathways, including superpathway of N-acetylneuraminate degradation, were related to liver and gut inflammation. The outcome of this study suggests a need for in-depth investigation of metabolic activity and other functions in the gut in future studies, as well as potential consideration of the role of sex in MC-LR toxicity.

Topics: Animals; Cyanobacteria; Feces; Female; Gastrointestinal Microbiome; Harmful Algal Bloom; Inflammation; Liver; Male; Marine Toxins; Metabolic Networks and Pathways; Mice; Microbiota; Microcystins; RNA, Ribosomal, 16S

Photocatalysis has been shown to successfully remove microcystins (MC) in laboratory experiments. Most research to date has been performed under ideal conditions in pure or ultrapure water. In this investigation the efficiency of photocatalysis using titanium dioxide was examined in a complex matrix (waste stabilisation lagoon water). A flow-through photocatalytic reactor was used for the photocatalytic removal of four commonly occurring microcystin analogues (MC-YR, MC-RR, MC-LR, and MC-LA). Up to 51% removal for single MC analogues in waste lagoon water was observed. Similar removal rates were observed when a mixture of all four MC analogues was treated. Although treatment of MC-containing cyanobacterial cells of Microcystis aeruginosa resulted in no decline in cell numbers or viability with the current reactor design and treatment regime, the photocatalytic treatment did improve the overall quality of waste lagoon water. This study demonstrates that despite the presence of natural organic matter the microcystins could be successfully degraded in a complex environmental matrix.

Topics: Cyanobacteria; Marine Toxins; Microcystins; Microcystis; Titanium; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical

Microcystin-leucine arginine (MC-LR) is a potent liver toxin produced by freshwater cyanobacteria, also known as blue-green algae. While harmful algal blooms are increasing in frequency and severity worldwide, there is still no established method for the diagnosis and assessment of MC-LR induced liver damage. The guidelines for MC-LR safe exposure limits have been previously established based on healthy animal studies, however we have previously demonstrated that pre-existing non-alcoholic fatty liver disease (NAFLD) increases susceptiblity to the hepatotoxic effects of MC-LR. In this study, we sought to investigate the suitability of clinically used biomarkers of liver injury, specifically alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as potential diagnostic tools for liver damage induced by chronic low dose administration of MC-LR in the setting of pre-existing NAFLD. In our Leprdb/J mouse model of NAFLD, we found that while MC-LR induced significant histopathologic damage in the setting of NAFLD, gene expression of ALT and ALP failed to increase with MC-LR exposure. Serum ALT and ALP also failed to increase with MC-LR exposure, except for a moderate increase in ALP with the highest dose of MC-LR used (100 μg/kg). In HepG2 human liver epithelial cells, we observed that increasing MC-LR exposure levels do not lead to an increase in ALT or ALP gene expression, intracellular enzyme activity, or extracellular activity, despite a significant increase in MC-LR induced cytotoxicity. These findings demonstrate that ALT and ALP may be unsuitable as diagnostic biomarkers for MC-LR induced liver damage.

Topics: Alanine Transaminase; Animals; Chemical and Drug Induced Liver Injury; Cyanobacteria; Gene Expression; Harmful Algal Bloom; Humans; Liver; Marine Toxins; Mice; Microcystins; Non-alcoholic Fatty Liver Disease

The effects of cyanobacteria (Aphanizomenon flos-aquae (90%), Microcystis aeruginosa) and dense Elodea canadensis beds on the health endpoints of the amphipod Gmelinoides fasciatus and bivalve mollusc Unio pictorum were examined in mesocosms with simulated summer conditions (July-August 2018) in the environment of the Rybinsk Reservoir (Volga River Basin, Russia). Four treatments were conducted, including one control and three treatments with influencing factors, cyanobacteria and dense elodea beds (separately and combined). After 20 days of exposure, we evaluated the frequency of malformed and dead embryos in amphipods, heart rate (HR) and its recovery (HRR) after stress tests in molluscs as well as heat tolerance (critical thermal maximum or CTMax) in both amphipods and molluscs. The significant effect, such as elevated number of malformed embryos, was recorded after exposure with cyanobacteria (separately and combined with elodea) and presence of microcystins (MC) in water (0.17 μg/l, 40% of the most toxic MC-LR contribution). This study provided evidence that an elevated number (>5% of the total number per female) of malformed embryos in amphipods showed noticeable toxicity effects in the presence of cyanobacteria. The decreased oxygen under the influence of dense elodea beds led to a decrease in HR (and an increase in HRR) in molluscs. The notable effects on all studied biomarkers, embryo malformation frequency and heat tolerance in the amphipod G. fasciatus, as well as the heat tolerance and heart rate in the mollusc U. pictorum, were found when both factors (elodea and cyanobacteria) were combined. The applied endpoints could be further developed for environmental monitoring, but the obtained results support the importance of the combined use of several biomarkers and species, especially in the case of multi-factor environmental stress.

Topics: Amphipoda; Animals; Aphanizomenon; Biomarkers; Bivalvia; Cyanobacteria; Embryo, Nonmammalian; Environmental Monitoring; Heart Rate; Hydrocharitaceae; Marine Toxins; Microcystins; Microcystis; Russia; Water Pollutants, Chemical

Microcystins produced by several toxic cyanobacterial strains constitute an important problem for public health. Bacterial degradation of these hepatotoxins may play an important role in natural ecosystems, however the nature of the process is very poorly understood. The aim of our study was to investigate the possible interactions between cyanotoxin producers and degraders. Samples collected from 24 water bodies in western Poland were analysed to determine the chemo-physical parameters, phytoplankton content, bacterial community structure and microcystin-biodegradation potency. A redundancy analysis identified a positive correlation between the capacity of a community to degrade microcystin LR (MC-LR) and temperature, pH, chlorophyll a concentration and the abundance of MC-producers. The relative abundance of classes F38, TM7-3 and the order WCHB1-81c (Actinobacteria) was significantly higher in the lakes with MC-biodegradation potency. Some specific bacterial genera belonging to Acidobacteria, Chloroflexi, Gemmatimonadetes, Firmicutes and TM7 were closely correlated with the occurrence of Microcystis spp. Furthermore, the MC biodegradation process was connected with the same bacterial groups. Thus, our approach allowed us to provide a broader picture of some specific relations between microcystin producers and potential microcystin degraders. A more comprehensive analysis of the existing correlations may be helpful in our understanding of natural mechanisms of MC elimination using bacteria such as MC-degraders.

Topics: Bacterial Toxins; Biodegradation, Environmental; Chlorophyll A; Cyanobacteria; Ecosystem; Lakes; Marine Toxins; Microcystins; Poland; Temperature; Water Microbiology

The composite photocatalyst of precious metal loaded on BiOI (M/BiOI, M = Pt, Au, Ag) was prepared by photochemical deposition and used for the photocatalytic degradation of microcystins (MC-LR). The material was characterized by X-ray powder diffraction (XRD), field emission scanning electron microscopy (FESEM), ultraviolet visible (UV-vis) diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy (XPS), and photoluminescence spectra (PL). The effect of photodegradation of MC-LR and the possible mechanism were investigated. It turned out that, among precious metals of Pt, Au, and Ag, Ag had the most significant improvement for photocatalytic activity of BiOI and Au was the least. The Ag/BiOI catalyst was illuminated 2 h under the simulated visible-light condition with the optimal load ratio of Ag catalyst (1.0 wt%) and the 2-h illumination under simulated visible-light condition, the degradation rate of MC-LR was 61.26% ± 0.12%. In addition, through the experiment of trapping agent and the analysis of electron spin resonance (ESR), we could conclude that the main active species is O

Topics: Catalysis; Light; Marine Toxins; Microcystins; Microscopy, Electron, Scanning; Models, Chemical; Photoelectron Spectroscopy; Photolysis; Semiconductors; X-Ray Diffraction

Cyclin-dependent protein kinase 6 (CDK6) plays a pivotal role in the regulation of the cell cycle and cell proliferation in mammals, and disruption of its expression by various microRNAs has been implicated in the pathogenesis of multiple human cancers. In mammals, miR-34a acts as a downstream effector of p53, and thus indirectly targets Cdk6, abrogating its effects. However, no studies have been done so far to examine the mechanistic involvement of miR-34a in the silencing of cdk6 in fish. In the present study, we found that the cDNA sequence of whitefish cdk6 has a 3'UTR region that contains a binding site for miR-34a. Using a luciferase reporter assay, we demonstrated that whitefish cdk6 is a direct target of miR-34a in vitro. In order to confirm this relationship in vivo, we measured the miR-34a and cdk6 mRNA expression patterns in the liver of whitefish after short-term (8, 24, and 48 h) and long-term (14 and 28 days) exposure to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. In contrast to the in vitro findings, we noticed an up-regulation of miR-34a and cdk6 expression after long-term MC-LR treatment. While these results indicate that both, miR-34a and cdk6 are responsive to MC-LR treatment, they do not support the presence of a miR-34a:cdk6 mRNA regulatory pair in the MC-LR-challanged whitefish liver in vivo. On the other hand, our findings suggests that cell regulatory elements, partnering with either miR-34a or cdk6, are worthy of further screening to better understand the molecular mechanisms that underlie the physiological response of fish challenged with hepatotoxic environmental pollutants like microcystins.

Topics: 3' Untranslated Regions; Animals; Cell Cycle; Cell Line; Cell Proliferation; Cyclin-Dependent Kinase 6; DNA, Complementary; HEK293 Cells; Humans; Liver; Marine Toxins; Microcystins; MicroRNAs; RNA, Messenger; Salmonidae; Tumor Suppressor Protein p53; Up-Regulation

Irrigation of crop plants with microcystins (MCs) contaminated water could be a threat to human health via bioaccumulation. Despite the fact MCs bioaccumulation in crop plants is well documented, MCs depuration, as well as the mechanism involved remains unclear. The objectives of the present study were to investigate the bioaccumulation and depuration of microcystin-LR (MC-LR) in lettuce (Lactuca sativa L.) and spinach (Spinacia oleracea L.), as well as to explore the role of glutathione (GSH) biosynthesis in MC-LR depuration. The tested plants were irrigated with deionized water containing 10 μg L

Topics: Animals; Glutathione; Glutathione Reductase; Glutathione Transferase; Humans; Lactuca; Marine Toxins; Microcystins; Spinacia oleracea

Microcystin-LR (MCLR) is the most commonly encountered toxic microcystin variant. MCLR is usually present along with common surface water constituents such as inorganic ions and natural organic matter (NOM) which compete with MCLR for active sites during ion exchange (IX) process. Consequently, development of a multicomponent competitive model is essential for practical IX applications. This is critically important given that the NOM characteristics (charge density and molecular weight distribution) and inorganic ions concentrations are spatially variable and can change seasonally. In the present study, a systematic study was carried out into the multicomponent interactions of IX resin with inorganic ions and NOM during the MCLR removal process. This involved evaluation of MCLR removal in a single component system (i.e., MCLR only), a dual component system (MCLR and one other contaminant such as NOM), and a multiple component system (MCLR with NOM and different inorganic ions present in natural waters). A comprehensive understanding of the dynamic adsorption behavior showed that the experimental data for single component systems agree well with a Freundlich isotherm. For multicomponent interactions, the Equivalent Background Concentration (EBC) model which is derived from the Ideal Adsorption Solution Theory (IAST) provided the best correlation with the experimental data in natural waters. The concentrations of competing NOM and inorganic ions estimated by the EBC model were <10% of their initial concentrations. Sulphates are the most competitive inorganic ions followed by nitrates and bicarbonates and the multicomponent interactions could be well predicted by using the IAST-EBC model. However, the EBC model failed in the presence of higher molecular weight Suwannee River Humic Acid (SRHA) molecules due to neglecting of the pore blocking phenomenon. In the presence of higher molecular weight SRHA molecules, the Redlich-Peterson Isotherm (RP) model exhibited a better performance than the Sheindorf-Rebuhn-Sheintuch (SRS) and the EBC models.

Topics: Adsorption; Humic Substances; Ion Exchange; Marine Toxins; Microcystins; Molecular Weight; Nitrates; Sulfates; Water Pollutants, Chemical; Water Purification

In this report, we establish proof-of-principle demonstrating for the first time genetic engineering of a photoautotrophic microorganism for bioremediation of naturally occurring cyanotoxins. In model cyanobacterium Synechocystis sp. PCC 6803 we have heterologously expressed Sphingopyxis sp. USTB-05 microcystinase (MlrA) bearing a 23 amino acid N-terminus secretion peptide from native Synechocystis sp. PCC 6803 PilA (sll1694). The resultant whole cell biocatalyst displayed about 3 times higher activity against microcystin-LR compared to a native MlrA host (Sphingomonas sp. ACM 3962), normalized for optical density. In addition, MlrA activity was found to be almost entirely located in the cyanobacterial cytosolic fraction, despite the presence of the secretion tag, with crude cellular extracts showing MlrA activity comparable to extracts from MlrA expressing E. coli. Furthermore, despite approximately 9.4-fold higher initial MlrA activity of a whole cell E. coli biocatalyst, utilization of a photoautotrophic chassis resulted in prolonged stability of MlrA activity when cultured under semi-natural conditions (using lake water), with the heterologous MlrA biocatalytic activity of the E. coli culture disappearing after 4 days, while the cyanobacterial host displayed activity (3% of initial activity) after 9 days. In addition, the cyanobacterial cell density was maintained over the duration of this experiment while the cell density of the E. coli culture rapidly declined. Lastly, failure to establish a stable cyanobacterial isolate expressing native MlrA (without the N-terminus tag) via the strong cpcB560 promoter draws attention to the use of peptide tags to positively modulate expression of potentially toxic proteins.

Topics: Biodegradation, Environmental; Cyanobacteria; Escherichia coli; Escherichia coli Proteins; Marine Toxins; Microcystins; Sphingomonas

Osteoporosis or enhanced bone loss is one of the most commonly occurring bone conditions in the world, responsible for higher incidence of fractures leading to increased morbidity and mortality in adults. Bone loss is affected by various environmental factors including diet, age, drugs, toxins etc. Microcystins are toxins produced by cyanobacteria with microcystin-LR being the most abundantly found around the world effecting both human and animal health. The present study demonstrates that MC-LR treatment induces bone loss and impairs both trabecular and cortical bone microarchitecture along with decreasing the mineral density and heterogeneity of bones in mice. This effect of MC-LR was found due to its immunomodulatory effects on the host immune system, wherein MC-LR skews both T cell (CD4

Topics: Animals; Arginine; Bone and Bones; Cyanobacteria; Immunity; Leucine; Marine Toxins; Mice; Microcystins; Minerals; Toxicity Tests; Tumor Necrosis Factor-alpha

Topics: China; Cyanobacteria; Environmental Monitoring; Humans; Lactuca; Lakes; Marine Toxins; Microcystins; Plant Leaves; Risk Assessment; Soil

Microcystin-LR (MC-LR) is the most abundant toxicant among microcystin variants produced by cyanobacteria. MC-induced toxicity is broadly reported to pose a threat to aquatic animals and humans and has been associated with the dysfunction of some organs such as liver and kidney. However, MC-induced neurotoxicity has not been well characterized after long-term exposure. This study was designed to investigate the neurotoxic effects after chronic oral administration of MC-LR. In our trial, C57/BL6 mice received MC-LR at 0, 1, 5, 10, 20 and 40 μg/L in drinking water for twelve months. Our data demonstrated that mitochondrial DNA (mtDNA) damage was evident in the damaged neurons as a result of chronic exposure. Histopathological abnormalities and mtDNA damage were observed in the hippocampus and cerebral cortex. Furthermore, MC-LR exerted distinct effects on these two brain regions. The hippocampus was more susceptible to the treatment of MC-LR compared with the cerebral cortex. However, no strong relationships were observed between the genotoxic effects and exposure doses. In conclusion, this study has provided a mtDNA-related mechanism for underlying chronic neurotoxicity of MC-LR and suggested the presence of differential toxicant effects on the hippocampus and cerebral cortex.

Topics: Animals; Cerebral Cortex; Cyanobacteria; DNA Damage; DNA, Mitochondrial; Hippocampus; Humans; Liver; Male; Marine Toxins; Mice; Microcystins; Mitochondria

In this study, statistically designed experiments using response surface methodology were conducted on Microcystis aeruginosa. A central composite design response surface model was established to investigate the multiple effects of various physical and chemical factors (total nitrogen, total phosphorus, temperature, and light intensity) on algal density and extracellular organic matter. The results of the experiments reveal that nitrate and phosphate had significant interactive effects on algal density, both iron and light intensity had synergic effects on the production of microcystins (MC-LR) and extracellular polysaccharides (EPS), and light intensity and nitrite had clear interactive effects on EPS release. Results did not show significant interactive effects on extracellular dissolved organic carbon (DOC) production. The contribution of extracellular dissolved organic matter of Microcystis aeruginosa during the logarithmic phase was further identified using a three-dimensional excitation emission matrix (3-DEEM). This study contributes to our theoretical knowledge of the prediction and analysis of M. aeruginosa growth and extracellular organic matter production.

Topics: Iron; Light; Marine Toxins; Microcystins; Microcystis; Models, Statistical; Nitrogen; Phosphorus; Temperature

The prevalence of cyanobacterial bloom (Cyano-bloom) and hepatotoxic microcystin (MC) pollution caused by eutrophication poses serious problems to aquatic ecosystems and public health. However, conventional water treatment technologies are inefficient for removing cyanotoxins. In this study, the performance of microcosm constructed wetlands (CWs) in the removal of Cyano-bloom, microcystin-LR (MC-LR), and nutrients was investigated following repeated loading of pollutants. The effects of plant and bioaugmentation of selected MC-LR degrading bacteria on removal efficiency, degrading gene mlrA abundance, and bacterial community structure were examined. More than 90% of the MC-LR and chlorophyll-a was eliminated by CWs after 3 d of hydraulic retention time (HRT) without a lag phase. No significant differences between planted and unplanted CWs were found in the MC-LR and Cyano-bloom removal and mlrA gene abundance. Nevertheless, the plants improved nutrient removal to reduce eutrophication. Bioaugmentation markedly enhanced the degradation of MC-LR from 16.7 μg L

Topics: Biodegradation, Environmental; Cyanobacteria; Eutrophication; Marine Toxins; Microcystins; Microcystis; Water Purification; Wetlands

Microcystin (MC) is a hepatotoxin produced by various cyanobacteria during harmful algal blooms (HAB's) in freshwater environments. Advanced treatment methods can remove MC from drinking water, but are costly and do not address recreational water exposure and ecosystem health concerns. Here we investigate the feasibility of utilizing plastics as a MC-adsorbing material, for use in water resources used for recreation, agriculture, aquaculture and drinking water. Water containing 20 µg/L MC-LR was exposed to polypropylene (PP) plastic for a six-day period at varying temperatures (22, 37, 65°C). Water samples were then collected at 0, 1, 2, and 6 hour-intervals to examine short term treatment feasibility. Samples were also taken at 24 hours, 3 days, and 6 days to determine long-term treatment effectiveness. MC concentrations were analyzed using ELISA. Results showed a maximal reduction of nearly 70% of MC-LR after a 6-day treatment with PP at 65°C. Temperature enhanced MC-LR reduction over a 6-day period: 70% reduction at 65°C; 50% at 37°C; 38% at 22°C. We propose an inexpensive intervention strategy which can be deployed rapidly on-site in various source waters, including in resource-limited settings. During the high peak of HAB season, the strategy can be applied in source waters, alleviating water treatment burden for treatment plants, lowering treatment costs and reducing chemical usage.

Topics: Adsorption; Cost-Benefit Analysis; Cyanobacteria; Ecosystem; Harmful Algal Bloom; Humans; Marine Toxins; Microcystins; Plastics; Polypropylenes; Recycling; Waste Disposal Facilities; Water Purification

Bacterial community isolated from different units of a Drinking Water Treatment Plant (DWTP) including pre-ozonation unit (POU), the effluent-sludge mixture of the sedimentation unit (ESSU) and top-sand layer water sample from the filtration unit (TSFU) were acclimatized separately in the microcystin-leucine arginine (MC-LR)-rich environment to evaluate MC-LR biodegradation. Maximum biodegradation efficiency of 97.2 ± 8.7% was achieved by the acclimatized-TSFU bacterial community followed by 72.1 ± 6.4% and 86.2 ± 7.3% by acclimatized-POU and acclimatized-ESSU bacterial community, respectively. Likewise, the non-acclimatized bacterial community showed similar biodegradation efficiency of 71.1 ± 7.37%, 86.7 ± 3.19% and 94.35 ± 10.63% for TSFU, ESSU and POU, respectively, when compared to the acclimatized ones. However, the biodegradation rate increased 1.5-folds for acclimatized versus non-acclimatized conditions. The mass spectrometry studies on MC-LR degradation depicted hydrolytic linearization of cyclic MC-LR along with the formation of small peptide fragments including Adda molecule that is linked to the reduced toxicity (qualitative toxicity analysis). This was further confirmed quantitatively by using Rhizobium meliloti as a bioindicator. The acclimatized-TSFU bacterial community comprised of novel MC-LR degrading strains, Chryseobacterium sp. and Pseudomonas fragi as confirmed by 16S rRNA sequencing.

Topics: Bacteria; Biodegradation, Environmental; Drinking Water; Filtration; Marine Toxins; Mass Spectrometry; Microcystins; RNA, Ribosomal, 16S; Sewage; Water Purification

Microcystin-leucine arginine (MC-LR) causes serum testosterone declines and male reproductive disorders. However, the molecular mechanisms underlying the pathological changes are still unclear. In the present study, we aimed to investigate the toxic effects of MC-LR on gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Our results demonstrated that MC-LR could enter GnRH neurons and inhibit GnRH synthesis, resulting in the decrease of serum GnRH and testosterone levels. The inhibitory effects of MC-LR on GnRH synthesis were identified to be associated with activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB)/c-Fos signaling pathway. With miRNA microarray analyses, we found that miR-329-3p was down-regulated most dramatically in MC-LR-treated GT1-7 cells. We then further identified that miR-329-3p regulated PRKAR1A and PRKACB expression and thus influenced GnRH synthesis. This is the first study to explore the molecular mechanism underlying the inhibitory effects of MC-LR on GnRH synthesis in the hypothalamus. Our data have provided a new perspective in the development of diagnosis and treatment strategies for male infertility as a result of dysfunction of the hypothalamic-pituitary-gonadal axis.

Topics: Animals; Cell Line; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinases; Gonadotropin-Releasing Hormone; Hypothalamus; Marine Toxins; Mice; Microcystins; MicroRNAs; Neurons; Signal Transduction; Transcription, Genetic

Blooms of harmful cyanobacteria have been observed in various water bodies across the world and some of them can produce intracellular toxins, such as microcystins (MCs), which negatively impact aquatic organisms and human health. Iron participates significantly in cyanobacterial photosynthesis and is proposed to be linked to MC production. Here, the cyanobacteria

Topics: Chlorophyll; Cyanobacteria; Iron; Marine Toxins; Microcystins; Microcystis; Oxidation-Reduction; Photosynthesis

Climate change and human activities induce an increased frequency and intensity of cyanobacterial blooms which could release toxins to aquatic ecosystems. Zooplankton communities belong to the first affected organisms, but in tropical freshwater ecosystems, this issue has yet been poorly investigated. We tested two questions (i) if the tropical Daphnia lumholtzi is capable to develop tolerance to an ecologically relevant concentration of purified microcystin-LR and microcystins from cyanobacterial extract transferable to F1 and F2 generations? And (ii) would F1 and F2 generations recover if reared in toxin-free medium? To answer these questions, we conducted two full factorial mutigenerational experiments, in which D. lumholtzi was exposed to MC-LR and cyanobacterial extract at the concentration of 1 μg L

Topics: Animals; Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Daphnia; Drug Resistance; Female; Lakes; Marine Toxins; Microcystins; Reproduction; Tropical Climate

Algal toxin is a unique type of toxin generated with harmful algal blooms in water bodies. This phenomenon is worsened by eutrophication caused by excessive discharge of nutrients into surface water bodies. Since algal toxins are hard to remove after they enter the water treatment processes, an efficient method is required to inhibit the growth of algal cells, to settle the cells at the bottom of the water body and to removes the toxin from the water. We report an efficient way to prepare a novel nanohybrid material, i.e., magnetic nanosilicate platelet (MNSP), and its effects on the removal of microcystin toxins as well as the cells of Microcystis aeruginosa. MNSP was fabricated by a special treatment of a clay mineral, montmorillonite, and then its surface was decorated with magnetite nanoparticles by in situ synthesis. The nanohybrid can efficiently inhibit the growth of M. aeruginosa-a typical species that can generate one of the most notorious algal toxins, i.e., microcystins. Algal cells can be settled with minimal 500 ppm MNSP, and the turbidity can be reduced by more than 67%. The removal of microcystin-LR (MC-LR) was as high as 99.39% at an concentration of 100 ppm, while the pristine nanosilicate platelet could only remove 36.84% at the same dosage.

Topics: Harmful Algal Bloom; Marine Toxins; Microcystins; Microcystis; Water Microbiology; Water Purification

Microcystins (MCs), produced by toxic cyanobacterial blooms that appeared world wildly in eutrophication waters, have often caused fish illness and even massive death cases. Among at least 90 structural variants, microcystin-LR (MC-LR) is the most common and toxic variant. In order to better understand innate immune responses in fish disrupted by environmental concentrations of MC-LR, male zebrafish (Danio rerio) were exposed to 0, 0.3, 1, 3, 10 and 30 μg/L MC-LR for 30 d, and the changes in splenic pathology and immunological gene expression as well as serum immune parameters were studied. In the low concentration groups (0.3, 1 and 3 μg/L), zebrafish displayed splenic inflammatory changes including the formation of melano-macrophage centers and the increase of macrophage pseudopodia, remarkable elevation of serum C3 levels, and significantly upregulated expression of innate immune-related genes (c3b, lyz, il1β, tnfα and ifnγ). In contrast, high concentrations of MC-LR (10 and 30 μg/L) resulted in the degeneration of splenic lymphocytes and macrophages, and down-regulation of immune-related genes as well as significant decreases in the level of serum C3. Furthermore, significant increases in the activity of serum ACP and ALP suggested that high concentrations of MC-LR increased permeability of macrophage plasma membrane or cellular necrosis, and subsequently decreased innate immune function. Our findings illustrated that sub-chronic exposure of MC-LR has dualistic influences on fish innate immune system with inflammatory activation at low exposure concentrations but turned to immune inhibition with the increases of exposure concentration.

Topics: Animals; Environmental Exposure; Fishes; Gene Expression Regulation; Immunity, Innate; Immunomodulation; Male; Marine Toxins; Microcystins; Models, Animal; Spleen; Transcriptional Activation; Zebrafish

Blooms of toxic cyanobacteria in Algerian reservoirs represent a potential health problem, mainly from drinking water that supplies the local population of Ain Zada (Bordj Bou Arreridj). The objective of this study is to monitor, detect, and identify the existence of cyanobacteria and microcystins during blooming times. Samples were taken in 2013 from eight stations. The results show that three potentially toxic cyanobacterial genera with the species Planktothrix agardhii were dominant. Cyanobacterial biomass, phycocyanin (PC) concentrations, and microcystin (MC) concentrations were high in the surface layer and at 14 m depth; these values were also high in the treated water. On 11 May 2013, MC concentrations were 6.3 μg/L in MC-LR equivalent in the drinking water. This study shows for the first time the presence of cyanotoxins in raw and treated waters, highlighting that regular monitoring of cyanobacteria and cyanotoxins must be undertaken to avoid potential health problems.

Topics: Biomass; Cyanobacteria; Drinking Water; Environmental Monitoring; Marine Toxins; Microcystins; Water Purification

A bloom of the cyanobacteria, Microcystis aeruginosa occurred in the St. Lucie Estuary during the summer of 2016, stimulated by the release of waters from Lake Okeechobee. This cyanobacterium produces the microcystins, a suite of heptapeptide hepatotoxins. The toxin composition of the bloom was analyzed and was compared to an archived bloom sample from 2005. Microcystin-LR was the most abundant toxin with lesser amounts of microcystin variants. Nodularin, cylindrospermopsin and anatoxin-a were not detected.

Topics: Bacterial Toxins; Biomass; Estuaries; Florida; Harmful Algal Bloom; Marine Toxins; Microcystins; Microcystis; Saline Waters

Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change-related factors. In this study, a new RT-qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin-LR (10 μg L

Topics: ATP-Binding Cassette Transporters; Bacterial Toxins; Climate Change; Environment; Environmental Microbiology; Gene Expression Regulation, Bacterial; Hot Temperature; Marine Toxins; Microcystins; Microcystis; Real-Time Polymerase Chain Reaction; Stress, Physiological; Water Microbiology

Recent studies have documented that microcystins (MCs) have potential toxic effects on growth and reproduction in fish. However, no systematic data exist on whether MCs cause gonadal development retardation through disrupting the growth hormone/insulin-like growth factors (GH/IGFs) system. To this end, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30µg/L microcystin-LR (MC-LR) for 90 d until they reached sexual maturity. Life-cycle exposure to MC-LR caused delayed ovarian maturation and sperm development along with ultrapathological lesions in the brain and liver. Moreover, the retarded gonadal development was accompanied by an inhibition of the GH/IGFs system, which was characterized by significant decreases in the transcriptional levels of brain gh (males only), hepatic igf2a and igf2b as well as gonadal igf1 (males only), igf3 and igf2r. These findings for the first time point to the influence of MC-LR on fish gonadal development via the GH/IGFs system. Also, sex-differential impairments suggested that gonadal development of males is more vulnerable than that of female to MC-LR. Our results provide evidence that MC-LR at environmentally relevant concentrations is able to induce impairments on fish gonadal development.

Topics: Animals; Brain; Cyanobacteria; Environmental Exposure; Enzyme Inhibitors; Female; Gonads; Growth Hormone; Insulin-Like Growth Factor I; Life Cycle Stages; Liver; Male; Marine Toxins; Microcystins; Ovary; Reproduction; Somatomedins; Spermatozoa; Water Pollutants, Chemical; Zebrafish

Freshwater blooms of cyanobacteria (blue-green algae) in source waters are generally composed of several different strains with the capability to produce a variety of toxins. The major exposure routes for humans are direct contact with recreational waters and ingestion of drinking water not efficiently treated. The ultra high pressure liquid chromatography tandem mass spectrometry based analytical method presented here allows simultaneous analysis of 22 cyanotoxins from different toxin groups, including anatoxins, cylindrospermopsins, nodularin and microcystins in raw water and drinking water. The use of reference standards enables correct identification of toxins as well as precision of the quantification and due to matrix effects, recovery correction is required. The multi-toxin group method presented here, does not compromise sensitivity, despite the large number of analytes. The limit of quantification was set to 0.1 μg/L for 75% of the cyanotoxins in drinking water and 0.5 μg/L for all cyanotoxins in raw water, which is compliant with the WHO guidance value for microcystin-LR. The matrix effects experienced during analysis were reasonable for most analytes, considering the large volume injected into the mass spectrometer. The time of analysis, including lysing of cell bound toxins, is less than three hours. Furthermore, the method was tested in Swedish source waters and infiltration ponds resulting in evidence of presence of anatoxin, homo-anatoxin, cylindrospermopsin and several variants of microcystins for the first time in Sweden, proving its usefulness.

Topics: Alkaloids; Bacterial Toxins; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Cyanobacteria; Cyanobacteria Toxins; Drinking Water; Fresh Water; Humans; Marine Toxins; Microcystins; Sweden; Tandem Mass Spectrometry; Tropanes; Uracil

There are some different freshwater algae in Eutrophic reservoirs which bloom with specific environmental conditions, and some of them are cyanobacteria. In this investigation, we have cultivated microalgae present in natural water samples from a eutrophic reservoir. Variations in temperature and light were evaluated, as well as the competition among different green algae and cyanobacteria. There were three different freshwater algae growing together, Scenedesmus sp., Kirchneriella sp. and Microcystis aeruginosa, this cyanobacterium was the algae that reached the highest development and growth during the culture. While the algae grew, the concentration of toxin (microcystin-LR) increased until it reached the highest levels at 570 μg g

Topics: Chlorophyta; Cyanobacteria; Ecosystem; Eutrophication; Fresh Water; Marine Toxins; Microcystins; Microcystis; Photoperiod; Scenedesmus; Seasons; Temperature; Water Supply

Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode Caenorhabditis elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa-enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the upregulation of cyp- and the downregulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth, and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely, the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR.

Topics: Adsorption; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Hydrocarbons, Brominated; Marine Toxins; Microcystins; Microcystis; Transcriptome; Water Pollutants, Chemical

Adsorption of mixtures of cyanotoxins onto sediment as a dominant mechanism in the elimination of cyanotoxins from the aqueous phase has not been extensively investigated. The aim of this study was to investigate adsorption and desorption behavior of six microcystins including microcystin (MC)-LR, RR, YR, LY, LW and LF and cylindrospermopsin (CYN) on natural sediment. Freundlich and Langmuir isotherms could be fitted for MC-LR, RR, YR and CYN. Sorption kinetics showed immediate rapid adsorption for all cyanotoxins: CYN, MCLW and MCLF were adsorbed 72.6%, 56.7% and 55.3% respectively within 2 h. Results of desorption experiments demonstrated that less than 9% of cyanotoxins desorbed from sediment within 96 h. Adsorption of cyanotoxins onto three fractionated sediments particles, clay-silt (<75 μm), find sand (75-315 μm) and coarse sand (315-2000 μm) demonstrated that adsorption capacity of coarse sand fraction for all the tested cyanotoxins was less than 4% of the clay-silt fraction. Results of this study revealed that there is a potential for cyanotoxins to accumulate in the sediments of lakes, as well as in drinking water treatment plants. Monitoring programs must consider cyanotoxins in the particulate phase to avoid largely underestimating toxin concentrations following their release from blooms.

Topics: Alkaloids; Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Drinking Water; Geologic Sediments; Lakes; Marine Toxins; Microcystins; Uracil; Water Pollutants, Chemical; Water Purification

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 μg L(-1) for total microcystins.

Topics: Chromatography, High Pressure Liquid; Drinking Water; Marine Toxins; Microcystins; Peptides, Cyclic; Phenylbutyrates; Tandem Mass Spectrometry; Water Microbiology

Concentration of toxic cyanobacteria blooms on the downwind shore of high recreational amenity water bodies with largely increases the risk of exposure to cyanotoxins. In this study analysis of phytoplankton structure, cyanotoxin composition and concentration was performed on cyanobacteria scum material, high- and low-density bloom samples in the Curonian Lagoon. We found that the concentration of cyanotoxins in the scum material increased from ∼30 to ∼300-fold compared to bloom samples. In Microcystis aeruginosa dominated samples microcystin-LR was present at the highest concentration, while the dominance of Planktothrix agardhii was associated with the occurrence of dmMC-RR as the major microcystin variant. The toxicological potential of cyanobacterial scums in the Curonian Lagoon is emphasized, and management by removal of these scums is proposed.

Topics: Aphanizomenon; Cyanobacteria; Environmental Monitoring; Eutrophication; Lithuania; Marine Toxins; Microcystins; Microcystis; Phytoplankton; Russia

Despite the focus of most ecotoxicological studies on cyanobacteria on a select group of cyanotoxins, especially microcystins, a growing body of evidence points to the involvement of other cyanobacterial metabolites in deleterious health effects. In the present study, original, self-developed reporter gene-based cellular biosensors, detecting activation of the main human xenobiotic stress response pathways, PXR and NFkappaB, were applied to detect novel potentially toxic bioactivities in extracts from freshwater microcystin-producing cyanobacterial blooms. Crude and purified extracts from cyanobacteria containing varying levels of microcystins, and standard microcystin-LR were tested. Two cellular biosensor types applied in this study, called NHRTOX (detecting PXR activation) and OXIBIOS (detecting NFkappaB activation), successfully detected potentially toxic or immunomodulating bioactivities in cyanobacterial extracts. The level of biosensor activation was comparable to control cognate environmental toxins. Despite the fact that extracts were derived from microcystin-producing cyanobacterial blooms and contained active microcystins, biosensor-detected bioactivities were shown to be unrelated to microcystin levels. Experimental results suggest the involvement of environmental toxins (causing a response in NHRTOX) and lipopolysaccharides (LPS) or other cell wall components (causing a response in OXIBIOS) in the potentially harmful bioactivity of investigated extracts. These results demonstrate the need for further identification of cyanobacterial metabolites other than commonly studied cyanotoxins as sources of health risk, show the usefulness of cellular biosensors for this purpose and suggest a novel, more holistic approach to environmental monitoring.

Topics: Animals; Biosensing Techniques; Cell Line; Cell Line, Tumor; Cell Survival; Cyanobacteria; Ecotoxicology; Environmental Monitoring; Hep G2 Cells; Humans; Marine Toxins; Microcystins; Rabbits; Water Pollutants, Chemical

Presence of microcystin (MC), a predominant freshwater algal toxin and a suspected liver carcinogen, in Florida's freshwaters poses serious health threat to humans and aquatic species. Being recalcitrant to conventional physical and chemical water treatment methods, biological methods of MC removal is widely researched. Water samples collected from five sites of Lake Okeechobee (LO) frequently exposed to toxic Microcystis blooms were used as inoculum for enrichment with microcystin LR (MC-LR) supplied as sole C and N source. After 20 days incubation, MC levels were analyzed using high performance liquid chromatography (HPLC). A bacterial consortium consisting of two isolates DC7 and DC8 from the Indian Prairie Canal sample showed over 74% toxin degradation at the end of day 20. Optimal temperature requirement for biodegradation was identified and phosphorus levels did not affect the MC biodegradation. Based on 16S rRNA sequence similarity the isolate DC8 was found to have a match with Microbacterium sp. and the DC7 isolate with Rhizobium gallicum (AY972457).

Topics: Actinomycetales; Bacterial Toxins; Base Sequence; Biodegradation, Environmental; Chromatography, High Pressure Liquid; Florida; Fresh Water; Marine Toxins; Microbial Consortia; Microcystins; Microcystis; Molecular Sequence Data; Phosphorus; Phylogeny; Rhizobium; RNA, Bacterial; RNA, Ribosomal, 16S; Temperature; Water Microbiology

Microcystins (MCs) produced by cyanobacteria and their continuing "blooms" are a worldwide problem owing to the toxicity of microcystin-LR (MC-LR) to plants and animals. In the present study, we investigated membrane transport of MC-LR and its toxic effects on zebrafish embryos using fragmentation of embryos, scanning electron microscope (SEM), fluorescence microscopy, and toxic exposure tests. At a concentration < 0.04 mmol/l, MC-LR was predominantly adsorbed on outer membrane surface of embryos according to Langmuir isotherm. The absorption characteristics of MC-LR within the range from 0.05 to 0.4 mmol/l conformed to Freundlich isotherm model. At concentrations > 0.50 mmol/l MC-LR directly entered the cytoplasm via partition. Thinning and disruption of membranes was confirmed using SEM and fluorescence morphological observations. Exposure to different concentrations of MC-LR resulted in differences in membrane transport and toxicity characteristics. At low concentrations, more than 75% of the adsorbed MC-LR accumulated on the outer membrane surface and resulted in axial malformation, tail curving, and tail twisting. Increasing the concentration of MC-LR to between 0.05 and 0.4 mmol/l improved membrane transport and it was evident in cytoplasm of embryos, resulting in serious pericardial edema, hatching gland edema, hemagglutination, hemorrhage, and vacuolization. At > 0.50 mmol/l, more than 70% of the adsorbed MC-LR entered the cytoplasm and this was lethal to the embryos. The current research outlines a new method and mechanism for the transmembrane transport of large molecular weight organic compounds and could be important for studies concerning molecular toxicology.

Topics: Animals; Dose-Response Relationship, Drug; Edema; Electrolytes; Embryo, Nonmammalian; Embryonic Development; Female; Hemagglutination; Hydrogen-Ion Concentration; Liposomes; Male; Marine Toxins; Microcystins; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Protein Transport; Temperature; Zebrafish; Zygote

Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

Topics: Animals; Biological Transport; Cell Separation; Enzyme Inhibitors; Female; Gene Expression; Hepatocytes; Humans; Liver; Male; Marine Toxins; Microcystins; Oligonucleotides, Antisense; Oocytes; Organic Anion Transporters; Phalloidine; Skates, Fish; Species Specificity; Substrate Specificity; Xenopus laevis

The ability of specific strains of probiotic bacteria to remove the cyanobacterial peptide toxin microcystin-LR from aqueous solutions was assessed. Lactobacillus rhamnosus strains GG and LC-705, Bifidobacterium longum 46, Bifidobacterium lactis 420 and Bifidobacterium lactis Bb12 were shown to be the most effective in toxin removal among 11 tested strains. The highest removal percentage of microcystin-LR was 58.1%, observed with B. lactis Bb12 (toxin concentration 100 microg L(-1), 10(10) CFU mL(-1), 37 degrees C, 24 h). Freshly cultured bacteria were shown to be more efficient in microcystin removal than lyophilized or nonviable bacteria. Removal of microcystin-LR was shown to be dependent on both temperature and bacterial concentration. It is concluded that some of the tested strains have good potential in removing microcystins from aqueous solutions.

Topics: Bacterial Toxins; Bifidobacterium; Chromatography, High Pressure Liquid; Cyanobacteria Toxins; Lacticaseibacillus rhamnosus; Marine Toxins; Microcystins; Probiotics; Time Factors

Cyanobacterial toxins have adverse effects on both terrestrial and aquatic plants. Microcystins are cyclic heptapeptides and an important group of cyanotoxins. When lake water contaminated with cyanobacterial blooms is used for spray irrigation, these toxins can come in contact with agricultural plants. During the exposure to these toxins, reactive oxygen species can form. These reactive oxygen species have a strong reactivity and are able to interact with other cellular compounds (lipids, protein, and DNA). Plants have antioxidative systems that will limit the negative effects caused by reactive oxygen species. These systems consist of enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, and nonenzymatic substances, such as reduced glutathione or vitamins. The aim of the present study was to investigate the effects of cyanobacterial toxins (microcystins and anatoxin-a) and cyanobacterial cell-free crude extract on alfalfa (Medicago sativa) seedlings. Inhibition of germination and root growth was observed with toxin concentrations of 5.0 microg/L. Also, oxidative damage, such as lipid peroxidation, was detected after the exposure of alfalfa seedlings to the toxin. Reactive oxygen detoxifying enzymes were elevated, showing a marked response in alfalfa to oxidative stress caused by the exposure to cyanobacterial metabolites that might influence the growth and development of these plants negatively.

Topics: Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Germination; Herbicides; Marine Toxins; Medicago sativa; Microcystins; Oxidative Stress; Paraquat; Peptides, Cyclic; Seeds; Tropanes

Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K(m) values amounted to 7 +/- 3 microM for OATP1B1, 9 +/- 3 microM for OATP1B3, and 20 +/- 8 microM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.

Topics: Animals; Brain; Female; Humans; Liver; Microcystins; Organic Anion Transporters; Peptides, Cyclic; Rats; Xenopus laevis

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method has been developed to identify and quantify microcystins in fish liver and intestine. Microcystins (MCs) were extracted from 500 mg sample with methanol-water (85:25, v/v) and the extracts concentrated to 250 microl. The parameters were optimized by a full factorial 2(3) design. Neither laborious pre-treatment nor clean up were necessary. MCs were separated using conventional C18 column and an acetonitrile-acidified water (pH 3) gradient. Negative samples (without MCs) were discriminated by liquid chromatography diode array detection (LC/DAD). The limits of detection (LOD) and the limits of quantification (LOQ) resulted equal for MC-RR, MC-YR, and MC-LR and were 0.1 and 0.5 microg g(-1), respectively. MCs recoveries at three levels in spiked samples (0.5-3.0 microg g(-1)) were > 92%, with relative standards deviations (RSDs) < 16% for liver samples and > 68% with RSDs < 18% for intestine samples. The proposed method was applied to determine MC-LR in exposed fish to evaluate the bioaccumulation risk. The results showed the transference of MC-LR from cyanobacterial cells to fish tissues.

Topics: Animals; Bacterial Toxins; Chemical Fractionation; Chromatography, Liquid; Cyanobacteria; Intestines; Liver; Male; Marine Toxins; Microcystins; Peptides, Cyclic; Spectrometry, Mass, Electrospray Ionization; Tilapia

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 microg g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 microg g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 microg g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood>liver>muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters.

Topics: Animals; Bacterial Toxins; Carcinogens; Carps; Eutrophication; Feeding Behavior; Marine Toxins; Microcystins; Peptides, Cyclic; Phytoplankton; Toxicity Tests

A water bloom of Planktothrix (Oscillatoria) rubescens was observed in a drinking water reservoir in central Spain in 2003. Microcystins where analysed by LC/ESI-MS in 21 samples collected from this reservoir in five different days between March and May. A demethylated variant of microcystin-RR was identified as the major microcystin in most samples. Trace levels of microcystin-LR, -RR and -YR were detected in some samples. Four less common microcystins, with [M+H]+ ions at m/z 960, 981, 1045 and 1053, were also found. Total extracellular microcystin concentration varied from 0.010 to 19.126 microg l(-1). Furthermore, anabaenopeptins B and F as well as Oscillamide Y were also identified in these samples.

Topics: Bacterial Toxins; Chromatography, Liquid; Cyanobacteria; Marine Toxins; Microcystins; Molecular Structure; Peptides, Cyclic; Spain; Spectrometry, Mass, Electrospray Ionization; Water; Water Microbiology

A series of acyclic, truncated microcystin analogues, comprised of the dienic beta-amino acid (Adda) and up to four additional amino acids characteristic of the parent toxin, was synthesized and screened for activity as inhibitors of PP1 and PP2A. Despite a recent report to the contrary for a microcystin-derived tetrapeptide degradation product, none approaches the potency of microcystin itself.

Topics: Bacterial Toxins; Enzyme Inhibitors; Inhibitory Concentration 50; Isoenzymes; Marine Toxins; Microcystins; Oligopeptides; Peptides, Cyclic; Phosphoprotein Phosphatases; Structure-Activity Relationship

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.

Topics: Animals; Artemia; Bacterial Toxins; Cyanobacteria; Enzyme Inhibitors; Glutathione; Glutathione Transferase; Inactivation, Metabolic; Marine Toxins; Microcystins; Peptides, Cyclic

A series of greatly simplified microcystin analogues comprised only of Adda (the beta-amino acid common to the microcystins, nodularins, and motuporin,) and a single additional amino acid residue was synthesized and screened for inhibition of the protein phosphatases 1 and 2A. Several of the analogues were shown to be mid-nanomolar inhibitors of the enzymes.

Topics: Amino Acids; Bacterial Toxins; Enzyme Inhibitors; Inhibitory Concentration 50; Isoenzymes; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Structure-Activity Relationship

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

Topics: Cross Reactions; Cyanobacteria; Immunoassay; Marine Toxins; Microcystins; Peptide Library; Peptides; Peptides, Cyclic; Water

The cyanobacterial toxins microcystins are known to affect a number of processes in plant tissues, and their presence in water used for irrigation may have considerable impact on the growth and development of crop plants. In this study, two plant bioassays were employed to investigate the phytotoxic effects of microcystins. A plant tissue culture assay revealed that the growth and chlorophyll content of Solanum tuberosum L. cultures was inhibited at microcystin-LR concentrations of 0.005 and 0.05 microg x cm(-3), respectively. A previously developed bioassay was also employed to determine the effects of three commonly occurring microcystin variants on the growth of Synapis alba L. seedlings. Microcystins-LR, -RR, and -LF inhibited the growth of seedlings, with GI50 values of 1.9, 1.6 and 7.7 microg x ml(-1), respectively. The growth of Phaseolus vulgaris was also examined in the presence of microcystin-LR. The toxin was found to have little effect on growth for up to 18 days, but impaired the development of the roots of exposed plants, causing them to take up approximately 30% less growth medium than those grown in the absence of toxin. Microcystin was also detected in the tissues of exposed plants using a commercially available ELISA kit, suggesting that the uptake of these toxins by edible plants may have significant implications for human health.

Topics: Bacterial Toxins; Enzyme-Linked Immunosorbent Assay; Marine Toxins; Microcystins; Peptides, Cyclic; Plant Development; Plant Growth Regulators; Plants

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

Topics: Cyanobacteria; Immunoassay; Marine Toxins; Microcystins; Peptides, Cyclic; Water Pollutants; Water Pollution

The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.

Topics: Administration, Oral; Animals; Antibody Formation; Enzyme-Linked Immunosorbent Assay; Female; Immune Sera; Immunization; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Peptides, Cyclic; Rabbits

A reliable and sensitive method for determination of hepatotoxic microcystins in complicated matrices by frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) is described. Immonium ions of constituent amino acids, which were obtained together with molecular ion species by FAB mass spectral analysis of standard microcystins RR, YR, LR, and [D-Asp3] and [Dha7]microcystins LR using flow injection system composed of Frit-FAB probe, showed potential for reliable identification of microcystins by Frit-FAB LC/MS. Frit-FAB LC/MS using a microbore column provided not only the baseline separation of standard microcystins RR, YR, and LR but 200-fold higher sensitivity than that using conventional column. Furthermore, when a selected ion monitoring (SIM) technique was used, the detection limits of microcystins RR, YR, and LR were 300, 350, and 400 pg, respectively, at a signal-to-noise ratio of 5:1, and calibration curves of each microcystin showed a linear relationship from 2 ng to 50 ng. Finally, identification and quantitative analyses of microcystins in water samples were carried out.

Topics: Chromatography, Liquid; Cyanobacteria; Flow Injection Analysis; Fresh Water; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Spectrometry, Mass, Fast Atom Bombardment