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Kaarina Sivonen - One of the best experts on this subject based on the ideXlab platform.
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production of high amounts of hepatotoxin Nodularin and new protease inhibitors pseudospumigins by the brazilian benthic nostoc sp cena543
Frontiers in Microbiology, 2017Co-Authors: Jouni Jokela, Lassi Matti Petteri Heinila, Tânia K Shishido, Matti Wahlsten, David P Fewer, Marli Fatima Fiore, Hao Wang, Esa Haapaniemi, Perttu Permi, Kaarina SivonenAbstract:Nostoc is cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore Brazilian cyanobacteria for new bioactive compounds and their characterization. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from small, shallow, saline-alkaline lake in the Nhecolândia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of Nodularin-R. This is the first free-living Nostoc found that produce Nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also found and characterized pseudospumigins A-F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 μM after 1 h) in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic Nodularin has the potential to cause deaths of eukaryotic organisms.
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Allelopathy of Baltic Sea cyanobacteria: No evidence for the role of Nodularin
2016Co-Authors: Sanna Suikkanen, Jouni Jokela, Kaarina Sivonen, Markku ViitasaloAbstract:Extracts of Aphanizomenon flos-aquae and Nodularia spumigena, the two most common cyano-bacteria forming recurrent blooms in the Baltic Sea, decrease the abundance of some phytoplankton species via the release of allelopathic substances. We investigated how cell-free filtrates of the two cyanobacteria, as well as purified hepatotoxin Nodularin, produced by N. spumigena affected cell numbers, chlorophyll a content and 14CO2 uptake of the cryptophyte Rhodomonas sp. Both cyanobacterial filtrates significantly retarded the growth of Rhodomonas sp., A. flos-aquae filtrate up to 46%, whereas purified Nodularin showed no significant effect on any of the growth parameters of the cryptophyte. These results suggest that the allelopathic effect of N. spumigena is most probably due to metabolite(s) other than Nodularin, possibly acting via the damage of the target cells
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Insights into the Physiology and Ecology of the Brackish- Water-Adapted Cyanobacterium Nodularia spumigena CCY9414 Based on a Genome-Transcriptome Analysis
2016Co-Authors: Henk Bolhuis, Kaarina Sivonen, Birgitta Bergman, David P Fewer, Rehab El-shehawy, Matthias Kopf, Fabian Haas, Paul Hayes, Elke Dittmann, Dave J. ScanlanAbstract:Nodularia spumigena is a filamentous diazotrophic cyanobacterium that dominates the annual late summer cyanobacterial blooms in the Baltic Sea. But N. spumigena also is common in brackish water bodies worldwide, suggesting special adaptation allowing it to thrive at moderate salinities. A draft genome analysis of N. spumigena sp. CCY9414 yielded a single scaffold of 5,462,271 nucleotides in length on which genes for 5,294 proteins were annotated. A subsequent strand-specific transcriptome analysis identified more than 6,000 putative transcriptional start sites (TSS). Orphan TSSs located in intergenic regions led us to predict 764 non-coding RNAs, among them 70 copies of a possible retrotransposon and several potential RNA regulators, some of which are also present in other N2-fixing cyanobacteria. Approximately 4 % of the total coding capacity is devoted to the production of secondary metabolites, among them the potent hepatotoxin Nodularin, the linear spumigin and the cyclic nodulapeptin. The transcriptional complexity associated with genes involved in nitrogen fixation and heterocyst differentiation is considerably smaller compared to other Nostocales. In contrast, sophisticated systems exis
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Nodularin uptake and induction of oxidative stress in spinach (Spinachia oleracea).
Journal of plant physiology, 2010Co-Authors: Nina Lehtimäki, Kaarina Sivonen, Jouni Jokela, Matti Wahlsten, Sumathy Shunmugam, Dalton Carmel, Mika Keränen, Eva-mari Aro, Yagut Allahverdiyeva, Paula MuloAbstract:The bloom-forming cyanobacterium Nodularia spumigena produces toxic compounds, including Nodularin, which is known to have adverse effects on various organisms. We monitored the primary effects of Nodularin exposure on physiological parameters in Spinachia oleracea. We present the first evidence for the uptake of Nodularin by a terrestrial plant, and show that the exposure of spinach to cyanobacterial crude water extract from Nodularin-producing strain AV1 results in inhibition of growth and bleaching of the leaves. Despite drastic effects on phenotype and survival, Nodularin did not disturb the photosynthetic performance of plants or the structure of the photosynthetic machinery in the chloroplast thylakoid membrane. Nevertheless, the Nodularin-exposed plants suffered from oxidative stress, as evidenced by a high level of oxidative modifications targeted to various proteins, altered levels of enzymes involved in scavenging of reactive oxygen species (ROS), and increased levels of α-tocopherol, which is an important antioxidant. Moreover, the high level of cytochrome oxidase (COX II), a typical marker for mitochondrial respiratory protein complexes, suggests that the respiratory capacity is increased in the leaves of Nodularin-exposed plants. Actively respiring plant mitochondria, in turn, may produce ROS at high rates. Although the accumulation of ROS and induction of the ROS scavenging network enable the survival of the plant upon toxin exposure, the upregulation of the enzymatic defense system is likely to increase energetic costs, reducing growth and the ultimate fitness of the plants.
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expression of the Nodularin synthetase genes in the baltic sea bloom former cyanobacterium nodularia spumigena strain av1
FEMS Microbiology Ecology, 2008Co-Authors: Sara Jonasson, Kaarina Sivonen, Simina Vintila, Rehab ElshehawyAbstract:Cyanobacterial blooms in the Baltic Sea are a common phenomenon and are formed by the heterocystous, filamentous species Nodularia spumigena. The toxicity of these blooms is attributed to the hepatotoxin Nodularin, produced by N. spumigena. Little is known regarding the regulatory mechanisms or environmental signaling that control Nodularin production. Here we report the characterization of the transcriptional expression pattern of the Nodularin synthetase gene cluster (nda) during phosphate depletion, and nitrogen supplementation. Real-time PCR analysis of these genes revealed that while cells continuously expressed the nda cluster, the expression of all nda genes increased when cells were subjected to phosphate depletion, and decreased in the presence of ammonium. In contrast to the shifts in expression, the intracellular and extracellular Nodularin concentrations did not vary significantly during the treatments.
Jussi Meriluoto - One of the best experts on this subject based on the ideXlab platform.
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Non-competitive ELISA with broad specificity for microcystins and Nodularins
Advances in Oceanography and Limnology, 2017Co-Authors: Akter, Lisa Spoof, Markus Vehniäinen, Jussi Meriluoto, Urpo LamminmäkiAbstract:Simple and cost-effective methods with sufficient sensitivities for preliminary screening of cyanobacterial toxins are in high demand for assessing water quality and safety. We have recently developed a highly sensitive and rapid time-resolved fluorometry based non-competitive immunoassay for detection of microcystins and Nodularins. The assay is based on a synthetic broad-specific anti-immunocomplex antibody SA51D1 capable of recognizing the immunocomplex formed by a generic anti-Adda monoclonal antibody (mAb) bound to either microcystins or Nodularins. Using the same antibody pair, here we describe a very simple and cost-efficient non-competitive ELISA test for microcystins and Nodularins based on conventional alkaline phosphatase (AP) activity measurement. The recombinant SA51D1 single-chain fragment of antibody variable domain (scFv) was produced as a fusion with bacterial alkaline phosphatase in Escherichia coli . After one step affinity purification through His-tag, the scFv-AP fusion protein could directly be used in the assay. For the assay, toxin standard/sample, biotinylated anti-Adda mAb and the scFv-AP were incubated together for one hour on streptavidin-coated microtiter wells, washed and AP activity was then measured by incubating (1 h at 37 ˚C) with chromogenic substrate para-nitrophenylphosphate (pNPP). The assay was capable of detecting all the eleven tested toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, LA -LY, -LF -LW, -WR, and Nodularin-R) below WHO guide line value of 1 µg L -1 . The detection limit (based on blank+3SD response) for microcystin-LR was ~0.2 µg L -1 . The assay was verified using spiked (0.25 - 4 µg L -1 of microcystin-LR) tap, river and lake water samples with recoveries from 64 to 101%. The assay showed good correlation (r 2 >0.9) with four reference methods for its performance in detecting extracted intracellular microcystin/Nodularin from 17 natural surface water samples. The described easy-to-perform assay has a high potential to be used in resource-poor settings as quantitative measurements can be obtained using a simple ELISA reader or easy-to-interpret qualitative results by visual readout. Based on the non-competitive format, the assay does not need any chemical toxin conjugates and offers robustness as compared to the currently available competitive format assays.
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fast separation of microcystins and Nodularins on narrow bore reversed phase columns coupled to a conventional hplc system
Toxicon, 2010Co-Authors: Lisa Spoof, Milla-riina Neffling, Jussi MeriluotoAbstract:Abstract Six modern narrow-bore (50 mm length × 2–2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three Nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 μm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1–4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 μm particle-based or monolithic reversed-phase columns for routine use in microcystin and Nodularin HPLC analyses.
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production and sedimentation of peptide toxins Nodularin r and microcystin lr in the northern baltic sea
Environmental Pollution, 2009Co-Authors: Harri Kankaanpää, Olli Sjovall, Miikka Olin, Vesa O. Sipiä, Krister Karlsson, Maija Huttunen, Kirsi Hyvarinen, Laura Sneitz, Janne Harkonen, Jussi MeriluotoAbstract:This seven-year survey was primarily targeted to quantification of production of Nodularin-R (NOD-R), a cyclic pentapeptide hepatotoxin, in Baltic Sea cyanobacteria waterblooms. Additionally, NOD-R and microcystin-LR (MC-LR; a cyclic heptapeptide toxin) sedimentation rates and NOD-R sediment storage were estimated. NOD-R production (70-2450 microg m(-3); approximately 1 kg km(-2) per season) and sedimentation rates (particles; 0.03-5.7 microg m(-2)d(-1); approximately 0.3kg km(-2) per season) were highly variable over space and time. Cell numbers of Nodularia spumigena did not correlate with NOD-R quantities. Dissolved NOD-R comprised 57-100% of total NOD-R in the predominantly senescent, low-intensity phytoplankton blooms and seston. Unprecedentedly intensive MC-LR sedimentation (0.56 microg m(-2)d(-1)) occurred in 2004. Hepatotoxin sedimentation rates highly exceeded those of anthropogenic xenobiotics. NOD-R storage in surficial sediments was 0.4-20 microg kg(-1) ( approximately 0.1 kg km(-2)). Loss of NOD-R within the chain consisting of phytoplankton, seston and soft sediments seemed very effective.
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Cyanobacterial hepatotoxins, microcystins and Nodularins, in fresh and brackish waters of the Pomeranian Province, Northern Poland
Oceanological and Hydrobiological Studies, 2008Co-Authors: Hanna Mazur-marzec, Lisa Spoof, Justyna Kobos, M. Plinski, Jussi MeriluotoAbstract:Abstract Microcystins (MCs) and structurally related Nodularins (NODs) are hepatotoxic cyclic peptides produced by bloom-forming cyanobacteria. These toxins have been implicated in the deaths of wild and domestic animals as well as in incidents of human illness. Cyanobacterial toxins occurring in the fresh and brackish waters of the Pomeranian Province, northern Poland were characterized in this study. Water samples collected from seven lakes in August and September 2005 were analysed by high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) and protein phosphatase inhibition assay (PPIA). Cyanobacterial toxins present in field samples and in an isolated strain of Planktothrix agardhii were also characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In most of the fresh water samples MC-LR, MC-RR and MC-YR dominated. In the lakes where P. agardhii was most abundant demethylated microcystin variants tentatively identified as [D-Asp3]MC-LR, [D-Asp3]MC-YR and [D-Asp3]MC-RR, were found. Total concentrations of the toxins measured by HPLC ranged from 0.1 μg 1-1 to 305.4 μg 1-1. Nodularia spumigena bloom samples were collected from brackish waters of the Gulf of Gdańsk, southern Baltic, and LC-ISP-MS/MS of extract from these revealed the presence of two geometrical isomers of linear Nodularin and Nodularin variant with aspartic acid methyl ester [MeAsp1(OMe)]NOD.
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nucleotide excision repair impairment by Nodularin in cho cell lines due to ercc1 xpf inactivation
Toxicology Letters, 2008Co-Authors: Anna Lankoff, Lisa Spoof, Jussi Meriluoto, Andrzej Wojcik, J. Sochacki, A. Wegierek, L. VerschaeveAbstract:The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as Nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of Nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for Nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of Nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with Nodularin (1 μg/ml, 10 μg/ml and 20 μg/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG−), UV41 (XPF−) and UV20 (ERCC1−) exposed to 20 J/m2 UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with Nodularin (10 μg/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that Nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.
Harri Kankaanpää - One of the best experts on this subject based on the ideXlab platform.
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Oxidative stress response in the red alga Furcellaria lumbricalis (Huds.) Lamour. due to exposure and uptake of the cyanobacterial toxin Nodularin from Nodularia spumigena.
Harmful Algae, 2010Co-Authors: Stephan Pflugmacher, Miikka Olin, Harri KankaanpääAbstract:The phenomenon of cyanobacterial blooms in the Baltic Sea is getting very common during the last few years. These blooms mostly consist of Nodularia species which can be toxic, producing Nodularins. As some of these blooms are wind driven to the coastline, an influence of the aquatic flora, especially on the macroalgae is expected. One of these common macroalgae is the red alga Furcellaria lumbricalis, living on hard bottom substrates in the lower part of the phytobenthic zone. Uptake of Nodularin into red alga the generation of oxidative stress, respectively the antioxidative response is shown measuring the activity of several antioxidative enzymes. The uptake of Nodularins by the red alga is shown. The toxins caused oxidative stress in the algae and induced an antioxidative defence.
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production and sedimentation of peptide toxins Nodularin r and microcystin lr in the northern baltic sea
Environmental Pollution, 2009Co-Authors: Harri Kankaanpää, Olli Sjovall, Miikka Olin, Vesa O. Sipiä, Krister Karlsson, Maija Huttunen, Kirsi Hyvarinen, Laura Sneitz, Janne Harkonen, Jussi MeriluotoAbstract:This seven-year survey was primarily targeted to quantification of production of Nodularin-R (NOD-R), a cyclic pentapeptide hepatotoxin, in Baltic Sea cyanobacteria waterblooms. Additionally, NOD-R and microcystin-LR (MC-LR; a cyclic heptapeptide toxin) sedimentation rates and NOD-R sediment storage were estimated. NOD-R production (70-2450 microg m(-3); approximately 1 kg km(-2) per season) and sedimentation rates (particles; 0.03-5.7 microg m(-2)d(-1); approximately 0.3kg km(-2) per season) were highly variable over space and time. Cell numbers of Nodularia spumigena did not correlate with NOD-R quantities. Dissolved NOD-R comprised 57-100% of total NOD-R in the predominantly senescent, low-intensity phytoplankton blooms and seston. Unprecedentedly intensive MC-LR sedimentation (0.56 microg m(-2)d(-1)) occurred in 2004. Hepatotoxin sedimentation rates highly exceeded those of anthropogenic xenobiotics. NOD-R storage in surficial sediments was 0.4-20 microg kg(-1) ( approximately 0.1 kg km(-2)). Loss of NOD-R within the chain consisting of phytoplankton, seston and soft sediments seemed very effective.
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accumulation and depuration of cyanobacterial toxin Nodularin and biomarker responses in the mussel mytilus edulis
Chemosphere, 2007Co-Authors: Harri Kankaanpää, Olli Sjovall, Sari Leinio, Miikka Olin, Jussi Meriluoto, Kari K. LehtonenAbstract:Blue mussels (Mytilus edulis) were exposed to an extract made of natural cyanobacterial mixture containing toxic cyanobacterium Nodularia spumigena (70–110 lg Nodularin l � 1 , 24-h exposure followed by 144-h depuration period in clean water). Toxin concentration increased from initial 400 to 1100 mg kg � 1 after 24-h exposure, measured by liquid chromatography/mass spectrometry (LC/MS). Acetylcholinesterase activity (AChE), a biomarker of direct neurotoxic effects, showed inhibition after 12 and 24 h exposure but returned to control level during the depuration period. Catalase (CAT) activity, an indicator of oxidative stress, showed significantly elevated levels in exposed mussels but only 72 h after the end of the exposure. No change in the activity of glutathione-S-transferase (GST) involved in conjugation reactions could be observed. A gradual yet incomplete elimination of Nodularin (from 1100 to 600 mg kg � 1 ) was observed during the depuration period, and the tissue levels were 30% lower in clean water after 24 h. The observed increase in oxidative stress indicated by elevated CAT activity is likely connected to detoxification reactions leading to the production of reactive oxygen species, including an apparent time lag in this specific enzymatic defence response. That no change in GST activity was observed suggests that this enzyme is not significantly involved in the detoxification process of Nodularin-containing cyanobacterial extract in M. edulis. � 2007 Elsevier Ltd. All rights reserved.
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transfer of Nodularin to three spined stickleback gasterosteus aculeatus l herring clupea harengus l and salmon salmo salar l in the northern baltic sea
Ecotoxicology and Environmental Safety, 2007Co-Authors: Vesa O. Sipiä, Harri Kankaanpää, Heikki Peltonen, M Vinni, Jussi MeriluotoAbstract:Abstract Nodularin (NODLN) is a hepatotoxin produced by the cyanobacterium Nodularia spumigena, which occurs regularly in the Baltic Sea. The primary aim of this study was to study the transfer of NODLN to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus membras L.), and salmon (Salmo salar L.), which were caught from the northern Baltic Sea between August 2002 and August 2003. Liquid chromatography mass spectrometry (LC-MS) was used for NODLN analysis. NODLN was found in both herring (0–90 μg kg−1 dw) and three-spined sticklebacks samples (2.8–700 μg kg−1 dw). The recovery for the spiked stickleback samples in vitro was 28%. Only 1 salmon of a total of 10 contained a small amount of NODLN (10 μg kg−1 dw). However, the high concentrations in individual stickleback suggest that possible transfer to higher trophic levels deserves more research.
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Nodularin induces oxidative stress in the Baltic Sea brown alga Fucus vesiculosus (Phaeophyceae)
Marine environmental research, 2007Co-Authors: Stephan Pflugmacher, Miikka Olin, Harri KankaanpääAbstract:Abstract In the Baltic Sea regular, intensive cyanobacterial blooms rich in the cyanobacterium Nodularia spumigena occur during the summer season. N. spumigena is known to produce the cyclic pentapeptide Nodularin (NOD) in high concentrations. Marine macroalgae, together with sea-grass meadows, are an extremely important habitat for life in the sea. In addition to this, the decaying macroalgae substantially contribute to the substrate for the microbial loop in coastal food webs. Uptake of Nodularin into the brown macroalga Fucus vesiculosus was assessed using an ELISA technique resulting in an uptake of up to 45.1 μg kg−1 fresh weight (fw). Nodularin was also detected in the reproductive part of the algae (receptacle) at 14.1 μg kg−1 fw. The induction of oxidative stress in F. vesiculosus, after exposure to NOD, was also shown by monitoring cellular damage as changes in lipid peroxidation and the activation of antioxidative defence systems (antioxidative capacity, superoxide dismutase and soluble glutathione S-transferase).
Wayne W. Carmichael - One of the best experts on this subject based on the ideXlab platform.
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cyanobacteria and prawn farming in northern new south wales australia a case study on cyanobacteria diversity and hepatotoxin bioaccumulation
Toxicology and Applied Pharmacology, 2005Co-Authors: Harri Kankaanpää, Jon Holliday, Helge Schroder, Timothy J Goddard, Richard Von Fister, Wayne W. CarmichaelAbstract:Harmful cyanobacteria pose a hazard to aquatic ecosystems due to toxins (hepatotoxic microcystins, Nodularins, and cylindrospermopsin) they produce. The microcystins and Nodularins are potent toxins, which are also tumor promoters. The microcystins and Nodularins may accumulate into aquatic organisms and be transferred to higher trophic levels, and eventually affect vector animals and consumers. Prawn farming is a rapidly growing industry in Australia. Because information regarding effects of cyanobacteria at prawn farms was lacking, we examined diversity of cyanobacteria and toxin production plus bioaccumulation into black tiger prawns (Penaeus monodon) under both field (northern New South Wales, Australia, December 2001-April 2002) and laboratory conditions. Samples were analyzed for hepatotoxins using enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). The maximum density of cyanobacteria (1 x 10{sup 6} to 4 x 10{sup 6} cells/l) was reached in April. Cyanobacteria encountered were Oscillatoria sp. (up to 4 x 10{sup 6} cells/l), Pseudanabaena sp. (up to 1.8 x 10{sup 6} cells/l), Microcystis sp. (up to 3.5 x 10{sup 4} cells/l), and Aphanocapsa sp. (up to 2 x 10{sup 4} cells/l). An uncommon cyanobacterium, Romeria sp. (up to 2.2 x 10{sup 6} cells/l), was also observed. Contrasting earlier indications, toxic Nodularia spumigena was absent.more » Despite that both Oscillatoria sp. and Microcystis sp. are potentially hepatotoxic, hepatotoxin levels in phytoplankton samples remained low (up to 0.5-1.2 mg/kg dw; ELISA) in 2001-2002. ELISA was found suitable not only for phytoplankton but prawn tissues as well. Enzymatic pretreatment improved extractability of hepatotoxin from cyanobacteria (Nodularin from N. spumigena as an example), but did not generally increase toxin recovery from prawn hepatopancreas. There were slightly increasing hepatotoxin concentrations in prawn hepatopancreas (from 6-20 to 20-80 {mu}g/kg dw; ELISA) during the study. Hepatotoxin concentrations in surface sediment remained low (<5 {mu}g/kg dw; ELISA) throughout the study. Laboratory experiments indicated that prawn hepatopancreas, heart, and brain were primary organs for hepatotoxin bioaccumulation. Toxin concentration in other organs, including muscle, was less effective. Orally administered Nodularin levels in hepatopancreas rapidly decreased from initial 830 to 250 {mu}g/kg dw in 96 h. Similarly, concentration of microcystin-LR injected in prawns decreased from 130 to 30 {mu}g/kg dw (hepatopancreas) in 2 h. These results demonstrate that potential risks caused by cyanobacteria in prawn farming (farmers, prawns, and consumers) were not substantial in 2001-2002. Although prawns may act as vectors for toxin transfer, they did not accumulate alerting amounts of hepatotoxins and were able to effectively detoxify them. Because bloom toxicity may vary, low-frequency toxin monitoring is recommended.« less
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Cyanobacteria and prawn farming in northern New South Wales, Australia--a case study on cyanobacteria diversity and hepatotoxin bioaccumulation.
Toxicology and applied pharmacology, 2005Co-Authors: Harri Kankaanpää, Jon Holliday, Helge Schroder, Timothy J Goddard, Richard Von Fister, Wayne W. CarmichaelAbstract:Harmful cyanobacteria pose a hazard to aquatic ecosystems due to toxins (hepatotoxic microcystins, Nodularins, and cylindrospermopsin) they produce. The microcystins and Nodularins are potent toxins, which are also tumor promoters. The microcystins and Nodularins may accumulate into aquatic organisms and be transferred to higher trophic levels, and eventually affect vector animals and consumers. Prawn farming is a rapidly growing industry in Australia. Because information regarding effects of cyanobacteria at prawn farms was lacking, we examined diversity of cyanobacteria and toxin production plus bioaccumulation into black tiger prawns (Penaeus monodon) under both field (northern New South Wales, Australia, December 2001-April 2002) and laboratory conditions. Samples were analyzed for hepatotoxins using enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). The maximum density of cyanobacteria (1 x 10{sup 6} to 4 x 10{sup 6} cells/l) was reached in April. Cyanobacteria encountered were Oscillatoria sp. (up to 4 x 10{sup 6} cells/l), Pseudanabaena sp. (up to 1.8 x 10{sup 6} cells/l), Microcystis sp. (up to 3.5 x 10{sup 4} cells/l), and Aphanocapsa sp. (up to 2 x 10{sup 4} cells/l). An uncommon cyanobacterium, Romeria sp. (up to 2.2 x 10{sup 6} cells/l), was also observed. Contrasting earlier indications, toxic Nodularia spumigena was absent.more » Despite that both Oscillatoria sp. and Microcystis sp. are potentially hepatotoxic, hepatotoxin levels in phytoplankton samples remained low (up to 0.5-1.2 mg/kg dw; ELISA) in 2001-2002. ELISA was found suitable not only for phytoplankton but prawn tissues as well. Enzymatic pretreatment improved extractability of hepatotoxin from cyanobacteria (Nodularin from N. spumigena as an example), but did not generally increase toxin recovery from prawn hepatopancreas. There were slightly increasing hepatotoxin concentrations in prawn hepatopancreas (from 6-20 to 20-80 {mu}g/kg dw; ELISA) during the study. Hepatotoxin concentrations in surface sediment remained low (
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Detection of Nodularin in flounders and cod from the Baltic Sea.
Environmental toxicology, 2001Co-Authors: Vesa O. Sipiä, Wayne W. Carmichael, Harri Kankaanpää, Kirsti Lahti, Jussi MeriluotoAbstract:The brackish water cyanobacterium Nodularia spumigena regularly forms waterblooms in the Baltic Sea. Many N. spumigena strains can produce Nodularin, a hepatotoxic penta-peptide, which has caused several animal poisonings in the Baltic Sea area. To improve our understanding of Nodularin bioaccumulation in aquatic organisms this study measured Nodularin in flounder and cod caught from the Baltic Sea. Flounders were collected from the western Gulf of Finland in July 1996, September 1997, and September 1998, and from the Gulf of Bothnia in August 1997 and September 1998. Flounders were also collected from the coastal areas of Sweden in the Baltic Proper during September 1998. Cod were caught from the southern Baltic Sea in August 1998. Livers and muscles of the 1997 fish were isolated, extracted, and analysed for Nodularin using high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 1 (PP1) inhibition assay. Approximately 30–70 ng of Nodularin/g dry weight (maximum value 140 ng/g) were found in the liver tissue samples by ELISA and PP1 inhibition. These concentrations were below the detection limit of HPLC. PP1 assay showed inhibition also in muscle samples, but this may due to other compounds present in the muscle extracts rather than NODLN or due to matrix interference. The recovery of Nodularin from liver tissue with ELISA and PP1 assays was about 30%. Nodularin concentrations in samples are not corrected for recovery. Although the concentrations of Nodularin found in this study are low further studies of Nodularin are needed to assess possible bioaccumulation in brackish water food webs. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 121–126, 2001
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Immuno-gold localization of hepatotoxins in cyanobacterial cells
Archives of Microbiology, 1995Co-Authors: Wayne W. Carmichael, Iain MillerAbstract: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 (nontoxin-producing strain), except that M. aerginosa PCC 7820 cells has a sheath. The thickness of the sheath was about 12nm 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.
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use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and Nodularins
Toxicon, 1994Co-Authors: Wayne W. CarmichaelAbstract: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.
Geoffrey A Codd - One of the best experts on this subject based on the ideXlab platform.
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Performance assessment of a cylindrospermopsin ELISA with purified compounds and cyanobacterial extracts
Environmental Forensics, 2017Co-Authors: James S Metcalf, Fiona M Young, Geoffrey A CoddAbstract:ABSTRACTDetecting low amounts of cyanobacterial toxins (cyanotoxins) in water and clinical materials is useful to protect human health and to understand cyanotoxin intoxication events. Commonly-occurring cyanobacterial compounds and extracts of cyanotoxin-producing cyanobacteria were tested by a cylindrospermopsin ELISA, in comparison with HPLC-PDA. Other than the cylindrospermopsin variants, none of the purified cyanobacterial peptides tested showed cross-reactivity in the cylindrospermopsin ELISA up to 100 µg/L, with the exception of Nodularin-R. The cylindrospermopsin concentration in cylindrospermopsin-producing cyanobacteria showed good agreement between ELISA and HPLC-PDA. However, extracts of Microcystis, Planktothrix, Nodularia, and Chlorella showed ELISA positives with such non-cylindrospermopsin-producing organisms reporting concentrations greater than 1 µg/L. When cylindrospermopsins were present, the cylindrospermopsin ELISA yielded good quantification, but false positive results from non-cyli...
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Nodularin in feathers and liver of eiders (Somateria mollissima) caught from the western Gulf of Finland in June–September 2005
Harmful Algae, 2008Co-Authors: Vesa O. Sipiä, Jussi Meriluoto, James S Metcalf, Milla-riina Neffling, Sonja Nybom, Geoffrey A CoddAbstract:Abstract Nodularins are cyanobacterial hepatotoxins, which may cause intoxication at very low exposure levels. The Nodularin-producing cyanobacterium Nodularia spumigena usually forms massive blooms in much of the Baltic Sea during the summer season. Breast feathers and liver samples from common eider ( Somateria mollissima ) were analysed for Nodularins by liquid chromatography–mass spectrometry (LC–MS) and enzyme-linked immunosorbent assay (ELISA). Fifteen eiders from the western Gulf of Finland were caught by hunters between June and September 2005. Blue mussels ( Mytilus edulis ), a dietary component of the birds, were also obtained by diving near the same marine area and time as the collection of the ducks. Eider breast feathers contained 6–52 μg Nodularin-R (Nod-R)/kg dry weight (dw) by ELISA, and 8–43 μg Nod-R/kg dw by LC–MS. No Nodularia filaments were adhered to feather samples according to light microscopy assessment. Liver samples from the same individuals contained Nod-R between 3 and 48 μg/kg dw by LC–MS. Mussel samples from the area contained Nod-R at concentrations of 12–80 μg/kg dw by LC–MS. Analysis of bird feathers offers a facile and non-invasive means of assessing the exposure of birds to Nodularins.
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Nodularin in feathers and liver of eiders somateria mollissima caught from the western gulf of finland in june september 2005
Harmful Algae, 2008Co-Authors: Vesa O. Sipiä, Jussi Meriluoto, James S Metcalf, Milla-riina Neffling, Sonja Nybom, Geoffrey A CoddAbstract:Abstract Nodularins are cyanobacterial hepatotoxins, which may cause intoxication at very low exposure levels. The Nodularin-producing cyanobacterium Nodularia spumigena usually forms massive blooms in much of the Baltic Sea during the summer season. Breast feathers and liver samples from common eider ( Somateria mollissima ) were analysed for Nodularins by liquid chromatography–mass spectrometry (LC–MS) and enzyme-linked immunosorbent assay (ELISA). Fifteen eiders from the western Gulf of Finland were caught by hunters between June and September 2005. Blue mussels ( Mytilus edulis ), a dietary component of the birds, were also obtained by diving near the same marine area and time as the collection of the ducks. Eider breast feathers contained 6–52 μg Nodularin-R (Nod-R)/kg dry weight (dw) by ELISA, and 8–43 μg Nod-R/kg dw by LC–MS. No Nodularia filaments were adhered to feather samples according to light microscopy assessment. Liver samples from the same individuals contained Nod-R between 3 and 48 μg/kg dw by LC–MS. Mussel samples from the area contained Nod-R at concentrations of 12–80 μg/kg dw by LC–MS. Analysis of bird feathers offers a facile and non-invasive means of assessing the exposure of birds to Nodularins.
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analysis of Nodularin r in eider somateria mollissima roach rutilus rutilus l and flounder platichthys flesus l liver and muscle samples from the western gulf of finland northern baltic sea
Environmental Toxicology and Chemistry, 2006Co-Authors: Vesa O. Sipiä, Olli Sjovall, James S Metcalf, Geoffrey A Codd, Terhi Valtonen, Deborah L Barnaby, Mikael Kilpi, Olli Mustonen, Jussi MeriluotoAbstract:Nodularin (NODLN) is a cyanobacterial hepatotoxin that may cause toxic effects at very low exposure levels. The NODLN-producing cyanobacterium Nodularia spumigena forms massive blooms in the northern Baltic Sea, especially during the summer. We analyzed liver and muscle (edible meat) samples from common eider (Somateria mollissima), roach (Rutilus rutilus L.), and flounder (Platichthys flesus L.) for NODLN-R by liquid chromatography/mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). Thirty eiders, 11 roach, and 15 flounders were caught from the western Gulf of Finland between September 2002 and October 2004. Eiders from April to June 2003 were found dead. The majority of samples were analyzed by LC-MS and ELISA from the same sample extracts (water:methanol:n-butanol, 75:20:5, v:v:v). Nodularin was detected in 27 eiders, nine roach, and eight flounders. Eider liver samples contained NODLN up to approximately 200 μg/kg dry weight and muscle samples at approximately 20 μg/kg dry weight, roach liver samples 20 to 900 μg NODLN/kg dry weight and muscle samples 2 to 200 μg NODLN/kg dry weight, and flounder liver samples approximately 5 to 1,100 μg NODLN/kg dry weight and muscle samples up to 100 μg NODLN/kg dry weight. The NODLN concentrations found in individual muscle samples of flounders, eiders, and roach (1–200 μg NODLN/kg dry wt) indicate that screening and risk assessment of NODLN in Baltic Sea edible fish and wildlife are required for the protection of consumer's health.
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Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and Nodularins of cyanobacteria
Applied and environmental microbiology, 2001Co-Authors: James S Metcalf, S. G. Bell, Geoffrey A CoddAbstract:A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and Nodularin detection and analysis do not provide information on the toxicity of microcystin and/or Nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and Nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and Nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and Nodularins.