nodularin and microcystin

nodularin has been researched along with microcystin* in 55 studies

Reviews

9 review(s) available for nodularin and microcystin

ArticleYear
Toxicopathology induced by microcystins and nodularin: a histopathological review.
    Journal of environmental science and health. Part C, Environmental carcinogenesis & ecotoxicology reviews, 2015, Volume: 33, Issue:2

    Cyanobacteria are present in all aquatic ecosystems throughout the world. They are able to produce toxic secondary metabolites, and microcystins are those most frequently found. Research has displayed a negative influence of microcystins and closely related nodularin on fish, and various histopathological alterations have been observed in many organs of the exposed fish. The aim of this article is to summarize the present knowledge of the impact of microcystins and nodularin on the histology of fish. The observed negative effects of cyanotoxins indicate that cyanobacteria and their toxins are a relevant medical (due to irritation, acute poisoning, tumor promotion, and carcinogenesis), ecotoxicological, and economic problem that may affect both fish and fish consumers including humans.

    Topics: Animals; Carcinogenesis; Cyanobacteria; Fish Diseases; Microcystins; Peptides, Cyclic

2015
Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.
    Marine drugs, 2015, Oct-22, Volume: 13, Issue:10

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

    Topics: Animals; Apoptosis; Carcinogens; Cell Death; Enzyme Inhibitors; Humans; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Reactive Oxygen Species

2015
State of knowledge and concerns on cyanobacterial blooms and cyanotoxins.
    Environment international, 2013, Volume: 59

    Cyanobacteria are ubiquitous microorganisms considered as important contributors to the formation of Earth's atmosphere and nitrogen fixation. However, they are also frequently associated with toxic blooms. Indeed, the wide range of hepatotoxins, neurotoxins and dermatotoxins synthesized by these bacteria is a growing environmental and public health concern. This paper provides a state of the art on the occurrence and management of harmful cyanobacterial blooms in surface and drinking water, including economic impacts and research needs. Cyanobacterial blooms usually occur according to a combination of environmental factors e.g., nutrient concentration, water temperature, light intensity, salinity, water movement, stagnation and residence time, as well as several other variables. These environmental variables, in turn, have promoted the evolution and biosynthesis of strain-specific, gene-controlled metabolites (cyanotoxins) that are often harmful to aquatic and terrestrial life, including humans. Cyanotoxins are primarily produced intracellularly during the exponential growth phase. Release of toxins into water can occur during cell death or senescence but can also be due to evolutionary-derived or environmentally-mediated circumstances such as allelopathy or relatively sudden nutrient limitation. Consequently, when cyanobacterial blooms occur in drinking water resources, treatment has to remove both cyanobacteria (avoiding cell lysis and subsequent toxin release) and aqueous cyanotoxins previously released. Cells are usually removed with limited lysis by physical processes such as clarification or membrane filtration. However, aqueous toxins are usually removed by both physical retention, through adsorption on activated carbon or reverse osmosis, and chemical oxidation, through ozonation or chlorination. While the efficient oxidation of the more common cyanotoxins (microcystin, cylindrospermopsin, anatoxin and saxitoxin) has been extensively reported, the chemical and toxicological characterization of their by-products requires further investigation. In addition, future research should also investigate the removal of poorly considered cyanotoxins (β-methylamino-alanine, lyngbyatoxin or aplysiatoxin) as well as the economic impact of blooms.

    Topics: Alkaloids; Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Drinking Water; Environmental Monitoring; Fresh Water; Microcystins; Peptides, Cyclic; Uracil; Water Microbiology

2013
On the chemistry, toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin.
    Marine drugs, 2010, May-10, Volume: 8, Issue:5

    The cyanobacteria or "blue-green algae", as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites.

    Topics: Alkaloids; Animals; Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Genes, Bacterial; Marine Toxins; Microcystins; Peptides, Cyclic; Saxitoxin; Uracil

2010
Cyanobacterial toxins--occurrence, biosynthesis and impact on human affairs.
    Molecular nutrition & food research, 2006, Volume: 50, Issue:1

    Mass developments of cyanobacteria ("blue-green algae") in lakes and brackish waters have repeatedly led to serious concerns due to their frequent association with toxins. Among these are the widespread hepatotoxins microcystin (MC) and nodularin (NOD). Here, we give an overview about the ecostrategies of the diverse toxin-producing species and about the genes and enzymes that are involved in the biosynthesis of the cyclic peptides. We further summarize current knowledge about toxicological mechanisms of MC and NOD, including protein phosphatase inhibition, oxidative stress and their tumor-promoting capabilities. One biotransformation pathway for MC is described. Mechanisms of cyanobacterial neurotoxins (anatoxin-a, homanatoxin-a, and anatoxin-a(s)) are briefly explained. We highlight selected cases of human fatalities related to the toxins. A special focus is given to evident cases of contamination of food supplements with cyanobacterial toxins, and to the necessary precautions.

    Topics: Animals; Bacterial Toxins; Carcinogens; Chemical and Drug Induced Liver Injury; Cyanobacteria; Cyanobacteria Toxins; Dietary Supplements; Ecosystem; Food Contamination; Humans; Marine Toxins; Microcystins; Oxidative Stress; Peptides, Cyclic; Phosphoprotein Phosphatases; Tropanes

2006
Evaluation of methods for the isolation, detection and quantification of cyanobacterial hepatotoxins.
    Aquatic toxicology (Amsterdam, Netherlands), 2006, Jul-20, Volume: 78, Issue:4

    Cyanobacterial hepatotoxins such as microcystins and nodularins have been responsible for the poisoning of both animals and humans who ingest or come into contact with toxic blooms. They are extremely stable in water due to their stable chemical structure and can tolerate radical changes in water chemistry, including pH and salinity. Different methods for the extraction and detection of these compounds have been reported. Extraction methods utilizing both aqueous and organic solvent systems have been reported. The detection methods ranging from immunological or biochemical assays such as enzyme linked immunosorbent assays (ELISA) and enzyme activity assays, to chemicals methods such as high performance liquid chromatography (HPLC) and more sophisticated liquid chromatography-mass spectrometry (LC-MS) have been documented as well. We review some important aspects of cyanobacterial hepatotoxins and methods of analysis for these toxins.

    Topics: Animals; Bacterial Toxins; Chromatography, High Pressure Liquid; Cyanobacteria; Electrophoresis, Capillary; Environmental Exposure; Enzyme-Linked Immunosorbent Assay; Eutrophication; Gas Chromatography-Mass Spectrometry; Humans; Liver; Marine Toxins; Microcystins; Nuclear Magnetic Resonance, Biomolecular; Peptides, Cyclic; Water Microbiology

2006
Algal toxins as guidance to identify phosphoproteins with key roles in apoptotic cell death.
    Current pharmaceutical biotechnology, 2006, Volume: 7, Issue:3

    The protein phosphatase inhibiting toxins microcystin and nodularin act rapidly to induce apoptotic cell death. Their inhibitory effect on protein phosphatases 1 and 2A can be utilized as tools to understand the phosphorylation-dependent regulatory mechanism underlying the early stage of apoptosis. The incubation of freshly isolated hepatocytes with these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appears [Fladmark, K. E., Brustugun, O. T., Hovland, R., Boe, R., Gjertsen, B. T., Zhivotovsky, B. and Doskeland, S. O. (1999) Cell Death Differ. 6, 1099-108]. Proteins subjected to phosphorylation in this early phase of apoptosis may play key roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-induction by microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainly serine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins reduces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectrometric identification and characterization. We will here give an overview of our strategy for identification of low-abundance phosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.

    Topics: Animals; Apoptosis; Cyanobacteria; Electrophoresis, Gel, Two-Dimensional; Hepatocytes; Humans; Mass Spectrometry; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphoproteins

2006
The microcystins and nodularins: cyclic polypeptide inhibitors of PP1 and PP2A.
    Current medicinal chemistry, 2002, Volume: 9, Issue:22

    The serine/threonine phosphatases are inhibited by a variety of natural toxins, including the microcystins and nodularins. Progress in understanding the details of the biosynthetic origin and the binding of these compounds is discussed, as is the progress made in synthesizing the members of these families. Additionally, the work by several groups to either synthesize simplified analogues that are still potent, or introduce selectivity for PP1 over PP2A are discussed. Finally, the properties of a series of five new truncated analogues are examined.

    Topics: Humans; Inhibitory Concentration 50; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Structure-Activity Relationship

2002
[Algal toxins, inhibitors of serine/threonine phosphatases].
    Comptes rendus des seances de la Societe de biologie et de ses filiales, 1998, Volume: 192, Issue:3

    Under certain environmental conditions, marine and freshwater phytoplankton may produce phycotoxins inhibitors of serine/thréonine protein phosphatases 1, 2A and 3. In the marine environment, dinoflagellates produce fatty polyethers: okadaic acid and its derivatives, the dinophysistoxins, which accumulate in shellfish and can cause diarrhetic shellfish poisoning (DSP) when ingested. In freshwater, the toxins are microcystins and nodularin, 7 or 5 amino acid cyclic peptides and are hepatotoxic. These toxins have caused massive poisoning of wild animals or domestic livestock and now are a health threat for humans through use of drinking and recreation water. Moreover, all these toxins are potent tumor promoters but belong to a new class, different from the TPA class, because they do not act on Protein Kinase C. Although the mutagenicity Ames test responds negatively, several results show their genotoxic potential, and therefore they are a health hazard through chronic exposition to low doses. Finally, okadaic acid, through its easy penetration in all cellular types can be used as a tool to study mechanisms involved in protein phosphorylation/dephosphorylation processes.

    Topics: Animals; Carcinogens; Cyanobacteria; Digestive System; Dinoflagellida; Enzyme Inhibitors; Eukaryota; Humans; Microcystins; Mutagens; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Phytoplankton; Shellfish Poisoning

1998

Other Studies

46 other study(ies) available for nodularin and microcystin

ArticleYear
A Feasibility Study into the Production of a Mussel Matrix Reference Material for the Cyanobacterial Toxins Microcystins and Nodularins.
    Toxins, 2022, 12-30, Volume: 15, Issue:1

    Microcystins and nodularins, produced naturally by certain species of cyanobacteria, have been found to accumulate in aquatic foodstuffs such as fish and shellfish, resulting in a risk to the health of the seafood consumer. Monitoring of toxins in such organisms for risk management purposes requires the availability of certified matrix reference materials to aid method development, validation and routine quality assurance. This study consequently targeted the preparation of a mussel tissue reference material incurred with a range of microcystin analogues and nodularins. Nine targeted analogues were incorporated into the material as confirmed through liquid chromatography with tandem mass spectrometry (LC-MS/MS), with an additional 15 analogues detected using LC coupled to non-targeted high resolution mass spectrometry (LC-HRMS). Toxins in the reference material and additional source tissues were quantified using LC-MS/MS, two different enzyme-linked immunosorbent assay (ELISA) methods and with an oxidative-cleavage method quantifying 3-methoxy-2-methyl-4-phenylbutyric acid (MMPB). Correlations between the concentrations quantified using the different methods were variable, likely relating to differences in assay cross-reactivities and differences in the abilities of each method to detect bound toxins. A consensus concentration of total soluble toxins determined from the four independent test methods was 2425 ± 575 µg/kg wet weight. A mean 43 ± 9% of bound toxins were present in addition to the freely extractable soluble form (57 ± 9%). The reference material produced was homogenous and stable when stored in the freezer for six months without any post-production stabilization applied. Consequently, a cyanotoxin shellfish reference material has been produced which demonstrates the feasibility of developing certified seafood matrix reference materials for a large range of cyanotoxins and could provide a valuable future resource for cyanotoxin risk monitoring, management and mitigation.

    Topics: Animals; Bivalvia; Chromatography, Liquid; Cyanobacteria Toxins; Feasibility Studies; Microcystins; Shellfish; Tandem Mass Spectrometry

2022
A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET).
    Analytical and bioanalytical chemistry, 2021, Volume: 413, Issue:24

    Topics: Cyanobacteria; Fluorescence Resonance Energy Transfer; Immunoassay; Limit of Detection; Microcystins; Peptides, Cyclic; Water Pollutants, Chemical

2021
Measurement of Microcystin and Nodularin Activity in Human Urine by Immunocapture-Protein Phosphatase 2A Assay.
    Toxins, 2019, 12-13, Volume: 11, Issue:12

    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.

    Topics: Humans; Immunoassay; Microcystins; Peptides, Cyclic; Protein Phosphatase 2

2019
Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9.
    Marine drugs, 2018, Feb-06, Volume: 16, Issue:2

    Strain B-9, which has a 99% similarity to

    Topics: Amino Acids; Antifungal Agents; Biodegradation, Environmental; Enzyme Inhibitors; Esterases; Gram-Negative Bacteria; Microcystins; Mycotoxins; Peptide Hydrolases; Peptides, Cyclic; Sulfonamides; Water Microbiology

2018
Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water.
    Analytical and bioanalytical chemistry, 2016, Volume: 408, Issue:22

    Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

    Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Enzyme-Linked Immunosorbent Assay; Female; Immunoconjugates; Limit of Detection; Mice, Inbred BALB C; Microcystins; Peptides, Cyclic; Water; Water Pollutants, Chemical

2016
Bioaccumulation of hepatotoxins - a considerable risk in the Latvian environment.
    Environmental pollution (Barking, Essex : 1987), 2015, Volume: 196

    The Gulf of Riga, river Daugava and several interconnected lakes around the City of Riga, Latvia, form adynamic brackish-freshwater system favouring occurrence of toxic cyanobacteria. We examined bioaccumulation of microcystins and nodularin-R in aquatic organisms in Latvian lakes, the Gulf of Riga and west coast of open Baltic Sea in 2002-2007. The freshwater unionids accumulated toxins efficiently,followed by snails. In contrast, Dreissena polymorpha and most lake fishes (except roach) accumulated much less hepatotoxins. Significant nodularin-R concentrations were detected also in marine clams and flounders. No transfer of nodularin-R and microcystins between lake and brackish water systems took place. Lake mussels can transfer hepatotoxins to higher organisms, and also effectively remove toxins from the water column. Obvious health risks to aquatic organisms and humans are discussed.

    Topics: Animals; Bacterial Toxins; Baltic States; Bivalvia; Cyanobacteria; Environmental Monitoring; Fishes; Flounder; Fresh Water; Lakes; Latvia; Microcystins; Oceans and Seas; Peptides, Cyclic; Water Pollutants

2015
Photocatalytic degradation of eleven microcystin variants and nodularin by TiO2 coated glass microspheres.
    Journal of hazardous materials, 2015, Dec-30, Volume: 300

    Microcystins and nodularin are toxic cyanobacterial secondary metabolites produced by cyanobacteria that pose a threat to human health in drinking water. Conventional water treatment methods often fail to remove these toxins. Advanced oxidation processes such as TiO2 photocatalysis have been shown to effectively degrade these compounds. A particular issue that has limited the widespread application of TiO2 photocatalysis for water treatment has been the separation of the nanoparticulate powder from the treated water. A novel catalyst format, TiO2 coated hollow glass spheres (Photospheres™), is far more easily separated from treated water due to its buoyancy. This paper reports the photocatalytic degradation of eleven microcystin variants and nodularin in water using Photospheres™. It was found that the Photospheres™ successfully decomposed all compounds in 5 min or less. This was found to be comparable to the rate of degradation observed using a Degussa P25 material, which has been previously reported to be the most efficient TiO2 for photocatalytic degradation of microcystins in water. Furthermore, it was observed that the degree of initial catalyst adsorption of the cyanotoxins depended on the amino acid in the variable positions of the microcystin molecule. The fastest degradation (2 min) was observed for the hydrophobic variants (microcystin-LY, -LW, -LF). Suitability of UV-LEDs as an alternative low energy light source was also evaluated.

    Topics: Catalysis; Cyanobacteria; Glass; Microcystins; Microspheres; Particle Size; Peptides, Cyclic; Photochemical Processes; Spectrophotometry, Ultraviolet; Titanium; Water Pollutants, Chemical; Water Purification

2015
Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS).
    Toxicon : official journal of the International Society on Toxinology, 2015, Sep-15, Volume: 104

    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

2015
Selectivity and potency of microcystin congeners against OATP1B1 and OATP1B3 expressing cancer cells.
    PloS one, 2014, Volume: 9, Issue:3

    Microcystins are potent phosphatase inhibitors and cellular toxins. They require active transport by OATP1B1 and OATP1B3 transporters for uptake into human cells, and the high expression of these transporters in the liver accounts for their selective hepatic toxicity. Several human tumors have been shown to have high levels of expression of OATP1B3 but not OATP1B1, the main transporter in liver cells. We hypothesized that microcystin variants could be isolated that are transported preferentially by OATP1B3 relative to OATP1B1 to advance as anticancer agents with clinically tolerable hepatic toxicity. Microcystin variants have been isolated and tested for cytotoxicity in cancer cells stably transfected with OATP1B1 and OATP1B3 transporters. Microcystin variants with cytotoxic OATP1B1/OATP1B3 IC50 ratios that ranged between 0.2 and 32 were found, representing a 150-fold range in transporter selectivity. As microcystin structure has a significant impact on transporter selectivity, it is potentially possible to develop analogs with even more pronounced OATP1B3 selectivity and thus enable their development as anticancer drugs.

    Topics: Cell Line, Tumor; Humans; Inhibitory Concentration 50; Liver-Specific Organic Anion Transporter 1; Microcystins; Neoplasms; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Peptides, Cyclic; Solute Carrier Organic Anion Transporter Family Member 1B3

2014
Multihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin.
    Environmental science & technology, 2014, Jul-15, Volume: 48, Issue:14

    Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO's maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

    Topics: Animals; Antibodies; Chromatography, Liquid; Cross Reactions; Drinking Water; Enzyme-Linked Immunosorbent Assay; Haptens; Humans; Immune Sera; Mass Spectrometry; Microcystins; Microcystis; Peptides, Cyclic; Reference Standards; Water Supply

2014
Community composition, toxigenicity, and environmental conditions during a cyanobacterial bloom occurring along 1,100 kilometers of the Murray River.
    Applied and environmental microbiology, 2012, Volume: 78, Issue:1

    A cyanobacterial bloom impacted over 1,100 km of the Murray River, Australia, and its tributaries in 2009. Physicochemical conditions in the river were optimal to support a bloom at the time. The data suggest that at least three blooms occurred concurrently in different sections of the river, with each having a different community composition and associated cyanotoxin profile. Microscopic and genetic analyses suggested the presence of potentially toxic Anabaena circinalis, Microcystis flos-aquae, and Cylindrospermopsis raciborskii at many locations. Low concentrations of saxitoxins and cylindrospermopsin were detected in Anabaena and Cylindrospermopsis populations. A multiplex quantitative PCR was used, employing novel oligonucleotide primers and fluorescent TaqMan probes, to examine bloom toxigenicity. This single reaction method identified the presence of the major cyanotoxin-producing species present in these environmental samples and also quantified the various toxin biosynthesis genes. A large number of cells present throughout the bloom were not potential toxin producers or were present in numbers below the limit of detection of the assay and therefore not an immediate health risk. Potential toxin-producing cells, possessing the cylindrospermopsin biosynthesis gene (cyrA), predominated early in the bloom, while those possessing the saxitoxin biosynthesis gene (sxtA) were more common toward its decline. In this study, the concentrations of cyanotoxins measured via enzyme-linked immunosorbent assay (ELISA) correlated positively with the respective toxin gene copy numbers, indicating that the molecular method may be used as a proxy for bloom risk assessment.

    Topics: Alkaloids; Bacterial Toxins; Base Sequence; Biota; Cyanobacteria; Cyanobacteria Toxins; DNA, Bacterial; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Microcystins; Molecular Sequence Data; New South Wales; Peptides, Cyclic; Polymerase Chain Reaction; Rivers; Saxitoxin; Uracil; Victoria; Water Microbiology

2012
Nostocyclopeptide-M1: a potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1.
    Molecular pharmaceutics, 2011, Apr-04, Volume: 8, Issue:2

    We have isolated a novel cyanobacterial cyclic peptide (nostocyclopeptide M1; Ncp-M1) that blocks the hepatotoxic action of microcystin (MC) and nodularin (Nod). We show here that Ncp-M1 is nontoxic to primary hepatocytes in long-term culture. Ncp-M1 does not affect any known intracellular targets or pathways involved in MC action, like protein phosphatases, CaM-KII, or ROS-dependent cell death effectors. In support of this conclusion Ncp-M1 had no protective effect when microinjected into cells. Rather, the antitoxin effect was solely due to blocked hepatocyte uptake of MC and Nod. The hepatic uptake of MC and Nod is mainly via the closely related organic anion transporters OATP1B1 and OATP1B3, which also mediate hepatic transport of endogenous metabolites and hormones as well as drugs. OATP1B3 is also expressed in some aggressive cancers, where it confers apoptosis resistance. We show that Ncp-M1 inhibits transport through OATP1B3 and OATP1B1 expressed in HEK293 cells. The Ncp-M1 molecule has several nonproteinogenic amino acids and an imino bond, which hamper its synthesis. Moreover, a cyclic all L-amino acid heptapeptide analogue of Ncp-M1 also inhibits the OATP1B1/1B3 transporters, and with higher OATP1B3 preference than Ncp-M1 itself. The nontoxic Ncp-M1 and its synthetic cyclic peptide analogues thus provide new tools to probe the role of OATB1B1/1B3 mediated drug and metabolite transport in liver and cancer cells. They can also serve as scaffolds to design new, exopeptidase resistant OATP1B3-specific modulators.

    Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; HEK293 Cells; Hepatocytes; Humans; Liver-Specific Organic Anion Transporter 1; Microcystins; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Peptides, Cyclic; Phosphorylation; Rats; Solute Carrier Organic Anion Transporter Family Member 1B3

2011
Quantitative assessment of aerosolized cyanobacterial toxins at two New Zealand lakes.
    Journal of environmental monitoring : JEM, 2011, Volume: 13, Issue:6

    The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular protein phosphatases. Toxicity primarily evolves following ingestion of cyanobacterial material or toxins and results in liver and renal pathology. Ingestion is the main route of exposure in the World Health Organizations current risk assessment of nodularin and microcystins. Nasally applied microcystin appears to have a 10-fold higher availability and toxicity than orally ingested toxins, suggesting that aerosolized toxins could represent a major risk for human populations close to lakes with cyanobacterial blooms. In this study, nodularin and microcystin levels in aerosols were assessed using high and low volume air samplers for 4, 12 and 24 h periods at lakes Forsyth and Rotorua (South Island, New Zealand). These lakes were experiencing blooms of Nodularia spumigena and Microcystis sp., respectively. Using the high volume samplers up to 16.2 pg m(-3) of nodularin and 1.8 pg m(-3) of microcystins were detected in the air. Aerosolized nodularin and microcystins do not appear to represent an acute or chronic hazard to humans. The latter was concluded based on calculations using average human air intakes, the highest nodularin or microcystin concentrations measured in the air in this study, and assuming inhalatory toxicities comparable to toxicological data obtained following intraperitoneal applications in mice. However, as the toxin concentrations in the air were calculated over extended sampling periods, peak values may be underestimated. Aerosolized toxins should be considered when developing risk assessments particularly for lakeside populations and recreational users where inhalation of cyanotoxins may be a secondary exposure source to a primary oral exposure.

    Topics: Aerosols; Air Microbiology; Air Pollutants; Atmosphere; Cyanobacteria; Environmental Monitoring; Fresh Water; Inhalation Exposure; Microcystins; New Zealand; Peptides, Cyclic

2011
Molecular methods: chip assay and quantitative real-time PCR: in detecting hepatotoxic cyanobacteria.
    Methods in molecular biology (Clifton, N.J.), 2011, Volume: 739

    Cyanobacterial mass occurrences are widespread and often contain hepatotoxic, i.e. microcystin- and nodularin-producing, species. Nowadays, detection of microcystin (mcy) and nodularin synthetase (nda) genes is widely used for the recognition of toxic cyanobacterial strains in environmental water samples. Chip assay presented here combines ligation detection reaction and hybridization on a universal microarray to detect and identify the mcyE/ndaF genes of five cyanobacterial genera specifically and sensitively. Thus, one chip assay can reveal the co-occurrence of several hepatotoxin producers. The presented quantitative real-time PCR method is used for the detection of either microcystin-producing Anabaena or Microcystis. Determination of the mcyE-gene copy numbers allows the identification of the dominant producer genus in the sample.

    Topics: Anabaena; Bacterial Proteins; Bacterial Typing Techniques; DNA Primers; DNA, Bacterial; Gene Dosage; Genes, Bacterial; Microarray Analysis; Microcystins; Microcystis; Peptides, Cyclic; Polymerase Chain Reaction; RNA, Ribosomal, 16S

2011
Differentiation of microcystin, nodularin, and their component amino acids by drop-coating deposition Raman spectroscopy.
    Analytical chemistry, 2011, Dec-15, Volume: 83, Issue:24

    Raman spectra of microcystin-LR (MC-LR), MC-RR, MC-LA, MC-LF, MC-LY, MC-LW, MC-YR, and nodularin collected by drop-coating deposition Raman (DCDR) spectroscopy are sufficiently unique for variant identification. Amino acid spectra of L-phenylalanine, L-leucine, L-alanine, D-alanine, L-glutamic acid, L-arginine, L-tryptophan, L-tyrosine, and N-methyl-D-aspartic acid were collected in crystalline, DCDR, and aqueous forms to aid in cyanotoxin Raman peak assignments. Both peak ratio analysis and principal component analysis (PCA) properly classified 72 DCDR spectra belonging to the eight toxins. Loading plots for the first three principal components (PCs) most heavily weighted the peaks highlighted in the peak ratio analysis, specifically the 760 cm(-1) tryptophan peak, 853 cm(-1) tyrosine peak, and 1006 cm(-1) phenylalanine peak. Peak ratio analyses may be preferred under some circumstances because of the ease and speed with which the ratios can be computed, even by untrained lab technicians. A set of rules was created to mathematically classify toxins using the peak ratios. DCDR methods hold great potential for future application in routine monitoring because portable and hand-held Raman spectrometers are commercially available, DCDR spectra can be collected in seconds for biomolecule mixtures as well as samples containing impurities, and the method requires far fewer consumables than conventional cyanotoxin detection methods.

    Topics: Amino Acids; Microcystins; Peptides, Cyclic; Principal Component Analysis; Spectrum Analysis, Raman

2011
Assessment of microcystin purity using charged aerosol detection.
    Journal of chromatography. A, 2010, Aug-06, Volume: 1217, Issue:32

    The increasing occurrence of toxic cyanobacterial blooms has led to a requirement for robust monitoring strategies and whilst several validated procedures have been developed these can be limited by the lack of high quality calibration standards. High quality standards must have confirmation of identity, purity and concentration by multiple methods. One aspect, purity, is rarely addressed but is essential. This is the first evaluation of the charged aerosol detector (CAD) to determine the benefits of incorporating a universal detector for more accurate purity determination of these peptides. Microcystins were detected at 5-10 ng on the column using the CAD, providing comparable quantification limits to those obtained using traditional UV detection. Purity determination of test compounds that had been partially purified, had showed that highest purity was at 238 nm>UV TIC>ESI TIC>CAD indicating that increased impurities could be detected using the CAD thus providing a more accurate indication of compound quality. Compounds purified by preparative HPLC were shown to have relative purities between 97% and 99%, however, when evaluated by CAD this dropped to 90-94% supporting the multi-detector strategy as essential for production of high quality compounds.

    Topics: Aerosols; Chromatography, High Pressure Liquid; Linear Models; Microcystins; Peptides, Cyclic; Sensitivity and Specificity

2010
Biodegradation of microcystins and nodularin in freshwaters.
    Chemosphere, 2008, Volume: 73, Issue:8

    Microcystin-LR (MC-LR) was readily biodegraded on addition to six different water samples irrespective of their previous exposure to microcystins. Subsequent studies with water from three of these water bodies confirmed the degradation of MC-LR and also demonstrated the biodegradation of MC-LF, nodularin and mixture of microcystins and nodularin. Rates of degradation of MC-LR, MC-LF and NOD in individual water samples ranged from a half-life of 4 to 18d. Analysis by HPLC-PDA-ESI+ and MALDI MS/MS revealed novel intermediate degradation products of MC-LF and nodularin which included demethylation, hydrolysis, decarboxylation and condensation of the parent compound(s). Our study suggests a possible diversity of micro-organisms and/or pathways which has not been previously observed.

    Topics: Amino Acid Sequence; Fresh Water; Microcystins; Peptides, Cyclic

2008
Identification of hepatotoxin-producing cyanobacteria by DNA-chip.
    Environmental microbiology, 2008, Volume: 10, Issue:3

    We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena, Microcystis, Planktothrix, Nostoc and Nodularia. Genus-specific probe pairs were designed for the detection of the microcystin (mcyE) and nodularin synthetase genes (ndaF) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE/ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE/ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1-5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.

    Topics: Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Cyanobacteria; DNA Fingerprinting; DNA, Bacterial; Ecosystem; Fresh Water; Microcystins; Oligonucleotide Array Sequence Analysis; Peptides, Cyclic; Polymerase Chain Reaction

2008
Microcystins associated with Microcystis dominated blooms in the Southwest wetlands, Western Australia.
    Environmental toxicology, 2006, Volume: 21, Issue:2

    Potentially toxic cyanobacterial blooms are becoming common in the freshwater wetlands on the Swan Coastal Plain, Western Australia. During summer the dominant bloom-causing species belong to the genera Microcystis and Anabaena and to a lesser extent Aphanizomenon and Nodularia. Although toxic cyanobacteria have been recorded in the Swan-Canning and Peel-Harvey estuaries in Western Australia, very little is known about the blooms in the surrounding freshwater lakes. In this study, a total of 32 natural bloom samples representing 13 lakes were analyzed by HPLC for microcystin (MC)-LR, -RR, and -YR. Twenty-eight samples proved to be toxic. The highest total microcystin concentration ranged from 1645 to 8428.6 microg L(-1), and the lowest concentrations were less than 10 microg L(-1) with some below the detection limit (< 0.05 microg L(-1)). MC-LR (100%) was the predominant microcystin, followed by MC-YR (71.4%) and MC-RR (60.7%). The presence of a Nodularia spumigena bloom in the freshwater Lake Yangebup was associated with the detection of nodularins (1664 microg L(-1)). This is the first study to demonstrate the presence of microcystins and nodularins in urban lakes on the Swan Coastal Plain, Western Australia.

    Topics: Bacterial Toxins; Chromatography, High Pressure Liquid; Environmental Monitoring; Fresh Water; Geography; Microcystins; Microcystis; Nodularia; Peptides, Cyclic; Western Australia

2006
Quantitative LC-ESI-MS analyses of microcystins and nodularin-R in animal tissue--matrix effects and method validation.
    Environmental toxicology, 2005, Volume: 20, Issue:3

    The matrix effects and signal response in LC-MS analysis of six microcystins and nodularin-R were studied in mussels and liver samples from the common eider and rainbow trout. The instrumentation used in the study was a triple quadrupole MS with electrospray ionization. The results from the spiked tissue samples showed that both signal suppression and enhancement occurred. The recorded matrix effects were not severe; all studied toxins could be detected with sufficient limit of detection in all matrices. The results indicate, however, that matrix effects must be monitored for accurate quantification of microcystin and nodularin in tissue samples. Matrix effects can be studied with standard additions in the studied matrix, as was done in this study. Solid-phase extraction (SPE) resulted in a lower limit of detection compared to no cleanup in the sample preparation. SPE also prolonged the chromatographic stability. SPE cleanup is therefore strongly recommeded. Also described in this article are the chromatographic and mass spectrometric details of glutathione and cysteine conjugates, which are the detoxification products of the toxins. LC-MS analysis is suitable for detoxification studies of microcystins and nodularins. Cysteine conjugate was identified as the main detoxification product in a mussel sample that was exposed to toxic cyanobacteria in an aquarium.

    Topics: Animals; Bivalvia; Chromatography, Liquid; Cysteine; Ducks; Mass Spectrometry; Microcystins; Oncorhynchus mykiss; Peptides, Cyclic

2005
A high proportion of Baltic Sea benthic cyanobacterial isolates contain apoptogens able to induce rapid death of isolated rat hepatocytes.
    Toxicon : official journal of the International Society on Toxinology, 2005, Sep-01, Volume: 46, Issue:3

    To assess the potential hepatotoxicity of benthic cyanobacteria, we isolated 41 strains from the Baltic Sea. The bacteria were differentially extracted with solvents of decreasing polarity. The extracts were tested for ability to induce death of primary rat hepatocytes in suspension culture. Mainly morphological criteria were used to discriminate between cell death with apoptotic features (shrinkage, chromatin hypercondensation, budding) or necrotic features (swelling, loss of plasma membrane integrity). The 24 isolates containing hepatotoxic compounds were of the genus Anabaena. The non-toxic isolates were mainly Nostoc and Calothrix. The toxicity was not due to the known hepatotoxic cyanobacterial protein phosphatase inhibitors microcystin or nodularin, as demonstrated by lacking competition with microcystin for PP2A binding. Apoptotic cell death was rapid, being evident from 10 to 60 min after the addition of extract. Sometimes the initial apoptosis was followed by secondary necrosis. Three cyanobacterial extracts produced apoptosis with unusual cell morphology including actin rearrangements. It will be of interest to know if they contain substance(s) acting through novel death pathways. We conclude that benthic Anabaena cyanobacteria represent a rich source of apoptogenic toxins, presumably directed against competitors or predators in the aquatic environment, but obviously able also to induce cell death in mammalian parenchymal liver cells.

    Topics: Actins; Animals; Apoptosis; Bacterial Toxins; Baltic States; Cells, Cultured; Cyanobacteria; Cyanobacteria Toxins; Enzyme Inhibitors; Hepatocytes; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Binding; Rats; Seawater; Time Factors

2005
Paucibacter toxinivorans gen. nov., sp. nov., a bacterium that degrades cyclic cyanobacterial hepatotoxins microcystins and nodularin.
    International journal of systematic and evolutionary microbiology, 2005, Volume: 55, Issue:Pt 4

    Thirteen bacterial isolates from lake sediment, capable of degrading cyanobacterial hepatotoxins microcystins and nodularin, were characterized by phenotypic, genetic and genomic approaches. Cells of these isolates were Gram-negative, motile by means of a single polar flagellum, oxidase-positive, weakly catalase-positive and rod-shaped. According to phenotypic characteristics (carbon utilization, fatty acid and enzyme activity profiles), the G+C content of the genomic DNA (66.1-68.0 mol%) and 16S rRNA gene sequence analysis (98.9-100% similarity) the strains formed a single microdiverse genospecies that was most closely related to Roseateles depolymerans (95.7-96.3% 16S rRNA gene sequence similarity). The isolates assimilated only a few carbon sources. Of the 96 carbon sources tested, Tween 40 was the only one used by all strains. The strains were able to mineralize phosphorus from organic compounds, and they had strong leucine arylamidase and chymotrypsin activities. The cellular fatty acids identified from all strains were C(16:0) (9.8-19%) and C(17:1)omega7c (<1-5.8%). The other predominant fatty acids comprised three groups: summed feature 3 (<1-2.2%), which included C(14:0) 3-OH and C(16:1) iso I, summed feature 4 (54-62%), which included C(16:1)omega7c and C(15:0) iso OH, and summed feature 7 (8.5-28%), which included omega7c, omega9c and omega12t forms of C(18:1). A more detailed analysis of two strains indicated that C(16:1)omega7c was the main fatty acid. The phylogenetic and phenotypic features separating our strains from recognized bacteria support the creation of a novel genus and species, for which the name Paucibacter toxinivorans gen. nov., sp. nov. is proposed. The type strain is 2C20(T) (=DSM 16998(T)=HAMBI 2767(T)=VYH 193597(T)).

    Topics: Bacterial Toxins; Betaproteobacteria; Biodegradation, Environmental; Cyanobacteria; DNA, Bacterial; DNA, Ribosomal; Fresh Water; Genes, rRNA; Geologic Sediments; Microcystins; Molecular Sequence Data; Peptides, Cyclic; Phenotype; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Species Specificity

2005
Simple assay for analyzing five microcystins and nodularin in fish muscle tissue: hot water extraction followed by liquid chromatography-tandem mass spectrometry.
    Journal of agricultural and food chemistry, 2005, Aug-24, Volume: 53, Issue:17

    A simple, specific, and sensitive procedure for determining six cyanotoxins, that is, microcystins RR, LR, YR, LA, and LW and nodularin, in fish muscle tissue is presented. This method is based on the matrix solid-phase dispersion technique with heated water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissue by 4 mL of water acidified to pH 2 and heated at 80 degrees C. After acidification and filtration, 0.2 mL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, with at least two precursor ion > product ion transitions selected for each target compound. Analyte recovery ranged between 61 and 82% and was not substantially affected by either the analyte concentrations or the type of fish. The nonexcellent recovery of some of the microcystins was traced to binding of these compounds to protein phosphatases in fish tissue occurring during sample treatment. The existence of covalently bound microcystins in fish has been evidenced by several studies. Compared to an older sample preparation procedure, this one extracted larger amounts of the analytes in a simpler and much more rapid way. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 1.6 and 4.0 ng/g. The effects of temperature and volume of the extractant on the analyte recovery were studied.

    Topics: Animals; Carcinogens; Chromatography, Liquid; Fishes; Marine Toxins; Mass Spectrometry; Microcystins; Muscles; Peptides, Cyclic; Quality Control; Spectrometry, Mass, Electrospray Ionization

2005
Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry.
    Chemosphere, 2004, Volume: 55, Issue:10

    Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.

    Topics: Chromatography, Liquid; Cyanobacteria; Fresh Water; Ireland; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Spectrometry, Mass, Electrospray Ionization; Ultraviolet Rays

2004
Detection and analysis of the cyanobacterial peptide hepatotoxins microcystin and nodularin using SELDI-TOF mass spectrometry.
    Toxicon : official journal of the International Society on Toxinology, 2004, Volume: 44, Issue:5

    Surface-enhanced laser desorption ionization mass spectrometry (SELDI-TOFMS) was used to develop a new and useful method for determination and identification of the cyanobacterial (blue-green algae) toxins: microcystin and nodularin. The technique, combining chromatography and MS, enables microcystin/nodularin capture, purification, analysis, and processing from complex biological mixtures directly onto a hydrophobic chip. Factors affecting ion intensities, including matrix concentration and laser intensity, were investigated to optimize sensitivity of the method. Microcystins and nodularin were analyzed for femtomolar sensitivity (about 2.5 pg microcystin-LR in 2 microl water). Samples of blood sera and liver tissue were spiked with microcystin-LR and analyzed. The detection limit was 1 ng in 2 microl blood sera solution. Reactions of microcystins by compounds containing mercaptan groups, such as dithiothreitol, aminoethanethiol and protein phosphatase 1, were examined on the chip by mass spectrometry. Formation of the microcystin-dithiothreitol conjugate was used to confirm the target compounds. The MS/MS data obtained showed the presence of the microcystin conjugate. The reaction position of the toxin with target compound was confirmed by a series of MS/MS fragment ions. The protein profile of microcystins reacting with protein phosphatase 1 was also obtained from the SELDI-TOF mass spectra.

    Topics: Cyanobacteria; Dithiothreitol; Enzyme Inhibitors; Ethanolamines; Mass Spectrometry; Microcystins; Molecular Structure; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Spectrometry, Mass, Electrospray Ionization; Sulfhydryl Compounds

2004
Microcystin biosynthesis in planktothrix: genes, evolution, and manipulation.
    Journal of bacteriology, 2003, Volume: 185, Issue:2

    Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

    Topics: Amino Acid Sequence; Bacterial Proteins; Cyanobacteria; Evolution, Molecular; Gene Deletion; Gene Expression Regulation, Bacterial; Methyltransferases; Microcystins; Molecular Sequence Data; Multigene Family; Peptide Synthases; Peptides, Cyclic; Transformation, Bacterial

2003
Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina.
    Aquatic toxicology (Amsterdam, Netherlands), 2003, Feb-12, Volume: 62, Issue:3

    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

2003
Effect of Microcystis aeruginosa and Nodularia spumigena on survival of Eurytemora affinis and the embryonic and larval development of the Baltic herring Clupea harengus membras.
    Environmental toxicology, 2003, Volume: 18, Issue:4

    Laboratory experiments were carried out to investigate the effect of two strains of Microcystis aeruginosa and a strain of Nodularia spumigena on the survival of Eurytemora affinis (Copepoda) and on the embryonic and larval development of the Baltic spring-spawning herring Clupea harengus membras. The trials were made in water taken from Pärnu Bay, at a salinity of 3.7-5.1 psu, a constant temperature (15 degrees C +/- 1 degrees C in trials with Eurytemora and herring embryos; 18 degrees C +/- 2 degrees C with herring larvae), and an oxygen concentration of 8.8-10.4 ppm. The strains tested had a negative impact on the survival of Eurytemora, as well as on the embryonic development and hatching regime of the Baltic herring. In Eurytemora the response depended on the sex of the animals: the survival was clearly higher in females. In the embryonic stages of herring, the influence resulted in an increase in deviations from the normal pattern of development and a higher mortality. The impact of the strains on the larval development of herring was rather moderate.

    Topics: Animals; Baltic States; Copepoda; Cyanobacteria; Fishes; Microcystins; Microcystis; Oceans and Seas; Peptides, Cyclic

2003
Extraction of 15 microcystins and nodularin using immunoaffinity columns.
    Toxicon : official journal of the International Society on Toxinology, 2003, Volume: 42, Issue:6

    Microcystins (MCYSTs) were isolated from surface water using reusable immunoaffinity columns. Individual MCYST were determined by high performance liquid chromatography equipped with a photo-diode array detector (HPLC-PDA, 200-300 nm). Subsequent analysis of the samples by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) provided molecular weight information, which was used to tentatively identify individual MCYST variants for which standards were not available. Results obtained using immunoaffinity columns (IAC)-HPLC-PDA were compared to those obtained using solid phase extraction (SPE) Oasis HLB-HPLC-PDA. This is the first report of the extraction of 15 microcystins and nodularin using immunoaffinity columns. Whereas previous reports demonstrates the use of IAC for four microcystins, we found that IAC selectively extracted the following microcystins: MCYST-RR, [D-Asp3]MCYST-RR, MCYST-YR, MCYST-LR, 3 MCYST-LR variants, MCYST-AR, MCYST-FR, MCYST-WR, MCYST-LA, MCYST-LA variant, the less polar microcystins such as MCYST-LF, MCYST-LW and nodularin. The IAC extracts were free of interferences which enabled better detection and identification of MCYSTs. Based on the amount loaded to the cartridges, the method detection limit was 10-14 ng when using IAC and 25 ng for SPE of each MCYST-RR, MCYST-YR and MCYST-LR. Reproducibilities expressed as relative standard deviation were 6-10% for SPE and 4-17% for IAC.

    Topics: Antibodies; Bacterial Toxins; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cyanobacteria; Marine Toxins; Microcystins; Peptides, Cyclic; Reference Values; Water

2003
Screening for cyanobacterial hepatotoxins, microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography-electrospray ionisation mass spectrometry.
    Journal of chromatography. A, 2003, Dec-05, Volume: 1020, Issue:1

    Water samples taken from 93 freshwater and brackish water locations in Aland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography-mass spectrometry (LC-MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC-UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS-MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 microg l(-1) were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 microg l(-1). The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC-UV were generally in good agreement with the LC-MS SIR results.

    Topics: Chromatography, High Pressure Liquid; Cyanobacteria; Enzyme-Linked Immunosorbent Assay; Liver; Microcystins; Peptides, Cyclic; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Water Pollutants, Chemical

2003
Ca2+/calmodulin-dependent protein kinase II is required for microcystin-induced apoptosis.
    The Journal of biological chemistry, 2002, Jan-25, Volume: 277, Issue:4

    The potent natural toxins microcystin, nodularin, and okadaic acid act rapidly to induce apoptotic cell death. Here we show that the apoptosis correlates with protein phosphorylation events and can be blocked by protein kinase inhibitors directed against the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The inhibitors used comprised a battery of cell-permeable protein kinase antagonists and CaMKII-directed peptide inhibitors introduced by microinjection or enforced expression. Furthermore, apoptosis could be induced by enforced expression of active forms of CaMKII but not with inactive CaMKII. It is concluded that the apoptogenic toxins, presumably through their known ability to inhibit serine/threonine protein phosphatases, can cause CaMKII-dependent phosphorylation events leading to cell death.

    Topics: 3T3 Cells; Animals; Apoptosis; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cells, Cultured; COS Cells; Dose-Response Relationship, Drug; Ecdysterone; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Hepatocytes; Humans; Inhibitory Concentration 50; Ionophores; Male; Mice; Microcystins; Microscopy, Fluorescence; Okadaic Acid; Peptides, Cyclic; Phosphorylation; Plasmids; Rats; Rats, Wistar; Time Factors; Transfection

2002
Rapid separation of microcystins and nodularin using a monolithic silica C18 column.
    Journal of chromatography. A, 2002, Feb-22, Volume: 947, Issue:2

    A monolithic C18-bonded silica rod column (Merck Chromolith) was compared to particle-based C18 and amide C16 sorbents in the HPLC separation of eight microcystins and nodularin-R. Two gradient mobile phases of aqueous trifluoroacetic acid modified with acetonitrile or methanol, different flow-rates and different gradient lengths were tested. The performance of the Chromolith column measured as the resolution of some microcystin pairs, the selectivity, efficiency (peak width) and peak asymmetry equalled, or exceeded, the performance of traditional particle-based columns. The Chromolith column allowed a shortening of the total analysis time to 4.3 min with a flow-rate 4 ml min(-1).

    Topics: Chromatography, High Pressure Liquid; Microcystins; Peptides, Cyclic

2002
High-performance liquid chromatographic separation of microcystins and nodularin, cyanobacterial peptide toxins, on C18 and amide C16 sorbents.
    Journal of chromatography. A, 2001, Feb-16, Volume: 909, Issue:2

    Four C18 columns and a novel amide C16 column were assessed in the HPLC separation of eight microcystins and nodularin-R. Gradient mobile phases of acetonitrile combined with trifluoroacetic acid, formic acid or ammonium acetate were compared. Special attention was paid to the resolution of four possible coeluting microcystin pairs. Generally speaking, the acidic mobile phases were superior to the ammonium acetate-based mobile phase in terms of resolution and selectivity. The amide C16 column had the best overall performance and unique selectivity properties.

    Topics: Bacterial Toxins; Chromatography, High Pressure Liquid; Cyanobacteria; Mass Spectrometry; Microcystins; Peptides, Cyclic; Spectrophotometry, Ultraviolet

2001
Microcystin-induced down-regulation of lymphocyte functions through reduced IL-2 mRNA stability.
    Toxicology letters, 2001, May-31, Volume: 122, Issue:1

    Here we report that lymphocyte functions were down-regulated by cyanobacterial hepatotoxin microcystin. Treatment of three microcystin (MC) isotypes, MC-LR, MC-YR and nodularin, on B6C3F1 mouse splenocytes produced dose-dependent inhibition of in vitro polyclonal antibody response and lymphoproliferation to LPS. ConA-induced lymphoproliferative response was decreased by MC-YR and nodularin, but no significant effect was observed in the MC-LR treatment. Intraperitoneal administration of nodularin into B6C3F1 mice decreased humoral immune responses to sheep red blood cell (sRBC), and the inhibitory effect became severe when hepatic uptake of nodularin was blocked by rifampicin. Each MC 1 microM suppressed phorbol 12-myristate 13-acetate (PMA) plus ionomycin-induced IL-2 mRNA expression in splenocytes and thymocytes, but not in EL-4 mouse thymoma cells. To further characterize the mechanism for the reduced IL-2 mRNA level, IL-2 mRNA stability was measured using RT-PCR. Deprivation of PMA/ionomycin stimuli from activated splenocytes and blockade of new transcription resulted in destabilization of IL-2 mRNA, which was accelerated by MC treatment. These results demonstrated that MC down-regulated lymphocyte functions and the immunosuppression was mediated, at least in part, through decreased IL-2 mRNA stability.

    Topics: Animals; Antibody Formation; Bacterial Toxins; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Female; Interleukin-2; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Microcystins; Peptides, Cyclic; RNA Stability; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

2001
Determination of cyanobacterial hepatotoxins directly in water using a protein phosphatase inhibition assay.
    Water research, 2001, Volume: 35, Issue:13

    A colorimetric phosphatase inhibition assay using protein phosphatase 2A and p-nitrophenyl phosphate as substrate for determining cyanobacterial peptide hepatotoxins directly in water without sample preconcentration has been developed. The assay uses commercially available materials and is much more simple to use than similar procedures using radiolabelled substrates. It has similar sensitivity to the radiolabelled assays and, with a working range of around 0.2-1 microg/L, is able to determine these toxins at concentrations below the provisional World Health Organisation guideline of 1 microg/L for microcystin-LR. The method appears robust and not to be affected by the sample matrix apart from possibly some components of cellular material if present at very high levels in extracts of cyanobacterial material. It is not affected by the presence of low levels of methanol in sample extracts.

    Topics: Animals; Bacterial Toxins; Buffers; Chromatography, High Pressure Liquid; Colorimetry; Cyanobacteria; Cyanobacteria Toxins; Female; Inhibitory Concentration 50; Liver; Marine Toxins; Methanol; Mice; Mice, Inbred BALB C; Microcystins; Muscle, Skeletal; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 2; Rabbits; Sensitivity and Specificity

2001
Time-dependent accumulation of cyanobacterial hepatotoxins in flounders (Platichthys flesus) and mussels (Mytilus edulis) from the northern Baltic Sea.
    Environmental toxicology, 2001, Volume: 16, Issue:4

    There is only limited information about the accumulation of algal toxins in aquatic organisms in the Baltic Sea. In this study we measured total cyanobacterial hepatotoxin levels in blue mussel (Mytilus edulis) and flounderi (Platichthys flesus) tissues. Flounder were caught with gillnets from the western Gulf of Finland during July and August 1999. Blue mussels were collected from an enclosure at 3 m depth and from an artificial reef (wreck, 25-35 m depth) in the western Gulf of Finland between June and September 1999. Flounder liver and muscle samples and soft tissues of mussels were analyzed for the cyanobacterial hepatotoxins (nodularin, NODLN and/or microcystins, MCs) using an enzyme-linked immunosorbent assay (ELISA). Results showed a time-dependent accumulation of hepatotoxins in flounder and mussels. In flounder, the maximum concentration 399 +/- 5 (sd) ng NODLN or MC/g dry weight (dw) was found in the liver of specimens caught on 21 August 1999. No hepatotoxins were detected in muscle samples. The maximum concentration of 2150 ng +/- 60 (sd) ng hepatotoxin/g dw was found in the mussel soft tissues collected on 20 August 1999. Temporal NODLN or MC trends indicated depuration of cyanobacterial hepatotoxin from mussels at surface level and an increase in NODLN or MC concentrations in those from the sea bed. These studies showed that despite the low cyanobacteria cell numbers the cyanobacterial hepatotoxins can accumulate in flounder and mussels. This may allow the further transfer of cyanobacterial hepatotoxins in the food web.

    Topics: Animals; Bivalvia; Body Weight; Cyanobacteria; Enzyme-Linked Immunosorbent Assay; Flounder; Kinetics; Liver; Microcystins; Muscle, Skeletal; Peptides, Cyclic; Seasons; Tissue Distribution

2001
Congener-independent immunoassay for microcystins and nodularins.
    Environmental science & technology, 2001, Dec-15, Volume: 35, Issue:24

    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

2001
Generation of antibodies directed against the low-immunogenic peptide-toxins microcystin-LR/RR and nodularin.
    International journal of immunopharmacology, 2000, Volume: 22, Issue:5

    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

2000
Influence of microcystine-YR and nodularin on the activity of some glucosidases in mouse liver.
    Toxicology, 2000, May-05, Volume: 146, Issue:2-3

    Microcystine-YR (20 ug/kg bodyweight) and 8 ug/kg bodyweight of nodularin were intraperitoneally injected to 90 female Swiss mice. After 15, 30, 60 min and 24 h the changes were observed in the activity of some glucosidases in the complete cell homogenate and in the lysosomal, microsomal and cytosol fraction. Significant differences were connected with the time after administration of poison and with the cellular fraction. Nodularin induces the activity of beta-D-glucuronidase, alpha-glucosidase, lysosomal esterase and N-acetyl-glucosaminidase and influenced on the labilization of endoplasmatic reticulum membranes. Microcystine-YR inhibited the biosynthesis of glucosidases and revealed a destructive effect on membranes of lysosomes and endoplasmatic reticulum.

    Topics: Animals; Bacterial Toxins; Cyanobacteria; Cytosol; Female; Glucosidases; Liver; Lysosomes; Mice; Microcystins; Microsomes, Liver; Peptides, Cyclic

2000
Isolation and detection of microcystins and nodularins, cyanobacterial peptide hepatotoxins.
    Methods in molecular biology (Clifton, N.J.), 2000, Volume: 145

    Topics: Animals; Biological Assay; Carcinogens; Chromatography, High Pressure Liquid; Cyanobacteria; Decapoda; Enzyme-Linked Immunosorbent Assay; Mice; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Water Microbiology

2000
Modulation of human polymorphonuclear leukocyte adherence by cyanopeptide toxins.
    Environmental research, 2000, Volume: 84, Issue:1

    The oligopeptides microcystins and nodularins are the most common and abundant cyanotoxins present in diverse water systems. They cause different illnesses in animal and humans, sometimes leading to death, and are responsible for severe environmental problems. Here we demonstrate that both microcystin-LR and N. spumigena nodularin (Nod) significantly enhance the early spontaneous adherence of peripheral polymorphonuclear leukocytes (PMNs) over the concentration range 10(-11)-10(-9) M. However, neither of them affect significantly the late spontaneous adherence or the early or late PMN-stimulated adherence (when cells are treated with formyl-methionyl-leucyl-phenylalanine). Since PMN adherence is a key step in the immune response, our data clearly indicate for the first time the immunomodulatory capacity of cyanopeptide toxins. The low concentrations at which the adherence modulation occurs are similar to the physiological concentrations for natural mammalian peptide hormones. Such concentrations are well below those recommended by other authors and World Health Organization in terms of risk assessment as safe for drinking water (8x10(-10) to 10(-9) M).

    Topics: Adult; Analysis of Variance; Bacterial Toxins; Cell Adhesion; Cyanobacteria; Environmental Monitoring; Humans; Male; Microcystins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peptides, Cyclic; Water Pollutants

2000
Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.
    Natural toxins, 1999, Volume: 7, Issue:6

    Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

    Topics: Alkaloids; Animals; Bacterial Toxins; Cyanobacteria Toxins; Enzyme-Linked Immunosorbent Assay; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Reagent Kits, Diagnostic; Sensitivity and Specificity; Uracil

1999
Assessment of rapid bioassays for detecting cyanobacterial toxicity.
    Letters in applied microbiology, 1995, Volume: 21, Issue:2

    Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato- and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3-17 mg l-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2-14 mg l-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.

    Topics: Animals; Artemia; Bacterial Toxins; Cyanobacteria; Cyanobacteria Toxins; Marine Toxins; Microcystins; Peptides, Cyclic; Photobacterium; Pseudomonas putida; Tropanes; Vibrio

1995
Immuno-gold localization of hepatotoxins in cyanobacterial cells.
    Archives of microbiology, 1995, Volume: 163, Issue:1

    A polyclonal antibody against the potent hepatotoxic cyclic peptide microcystins and nodularins was used in conjunction with immuno-gold labelling to localize the toxins in three strains of cyanobacteria. Ultrastructurally, there were no major differences between unicellular Microcystis aeruginosa strain PCC 7820 (toxin-producing strain) and M. aeruginosa strain UTEX 2063 (non-toxin-producing strain), except that M. aeruginosa PCC 7820 cells had a sheath. The thickness of the sheath was about 12 nm and was distinguishable from the cell wall at the ultrastructural level only when the specimen was stained en bloc with uranyl acetate. Microcystins and nodularin were found in M. aeruginosa PCC 7820 and Nodularia spumigena strain L-575 respectively, but not in nontoxic M. aeroginosa UTEX 2063. In M. aeruginosa PCC 7820 cells, microcystin was found primarily in the thylakoid area and nucleoid, with smaller amounts in the cell wall and sheath. Only nonspecific labelling was found in other cellular inclusions, such as polyhedral bodies, cyanophycin granules and membrane-limited inclusions. In strain N. spumigena L-575, nodularin was found in both vegetative cells and heterocysts with a distribution similar to that in M. aeruginosa PCC 7820.

    Topics: Bacterial Toxins; Cell Membrane; Cell Nucleus; Cell Wall; Cyanobacteria; Immunohistochemistry; Microcystins; Microscopy, Electron; Peptides, Cyclic

1995
Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins.
    Toxicon : official journal of the International Society on Toxinology, 1994, Volume: 32, Issue:12

    Microcystins and nodularins are cyclic peptide hepatotoxins and tumor promoters produced by several genera of cyanobacteria. Using a rabbit anti-microcystin-LR polyclonal antibody preparation, the cross-reactivity with 18 microcystin and nodularin variants was tested. A hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which has the (E) form at the C-6 double bond in both microcystin and nodularin, was found essential for these toxins to express antibody specificity. Modification of -COOH in glutamic acid of microcystin and nodularin did not alter their antigenicity. Antibody cross-reactivity of these toxins was compared with their ability to inhibit protein phosphatase type 1 (PP1). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosphate activity toward PP1. PP1 was obtained as recombinant PP1 expressed in E. coli. The inhibition effect of five microcystins and two nodularins on recombinant PP1 activity toward p-nitrophenol phospate was measured in a microwell plate reader. The concentration of microcystin-LR causing 50% inhibition of recombinant PP1 activity (IC50) was about 0.3 nM, while that of two modified microcystins had a significantly higher IC50. Microcystin-LR and nodularin with the (z) form of Adda at the C-6 double bond or having the monoester of glutamic acid did not inhibit PP1. These three toxins were also nontoxic in the mouse bioassay. These results show the importance of Adda and glutamic acid in toxicity of these cyclic peptides and that PP1 inhibition is related to the toxins' mechanism of action.

    Topics: Amino Acids; Animals; Antibodies; Antibody Specificity; Bacterial Toxins; Binding, Competitive; Colorimetry; Cross Reactions; Cyanobacteria; Enzyme-Linked Immunosorbent Assay; Microcystins; Molecular Weight; Peptides, Cyclic; Phosphoprotein Phosphatases; Rabbits; Recombinant Proteins; Reference Standards

1994
Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria.
    Natural toxins, 1994, Volume: 2, Issue:4

    Bacterial degradation of the cyanobacterial cyclic peptide hepatotoxin microcystin was confirmed in natural waters and by isolated laboratory strains. Degradation of 1 mg L-1 microcystin LR typically began 2-8 days after addition to surface water samples. At concentrations greater than 1 mg L-1 there was an initial slow removal of microcystin LR, rather than a distinct lag (or conditioning) phase, before rapid degradation commenced. The lag phase was absent upon re-addition of microcystin LR to the water. Both single strains and mixed bacterial cultures capable of degrading microcystin LR were isolated from surface water samples. One single strain isolated was a gram-negative rod and appeared to be a Pseudomonas sp., although standard taxonomic tests have given inconclusive results. Degradative activity was mostly intracellular and equally active against microcystin LR and RR, but not against nodularin.

    Topics: Biodegradation, Environmental; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases

1994