Vitor Vasconcelos

Molecular Mechanisms of Microcystin Toxicity in Animal Cells

Microcystins (MC) are potent hepatotoxins produced by the cyanobacteria of the genera Planktothrix, Microcystis, Aphanizomenon, Nostoc and Anabaena. These cyclic heptapeptides have strong affinity to serine/threonine protein phosphatases (PPs) thereby acting as an inhibitor of this group of enzymes. Through this interaction a cascade of events responsible for the MC cytotoxic and genotoxic effects in animal cells may take place. Moreover MC induces oxidative stress in animal cells and together with the inhibition of PPs, this pathway is considered to be one of the main mechanisms of MC toxicity. In recent years new insights on the key enzymes involved in the signal-transduction and toxicity have been reported demonstrating the complexity of the interaction of these toxins with animal cells. Key proteins involved in MC up-take, biotransformation and excretion have been identified, demonstrating the ability of aquatic animals to metabolize and excrete the toxin. MC have shown to interact with the mitochondria. The consequences are the dysfunction of the organelle, induction of reactive oxygen species (ROS) and cell apoptosis. MC activity leads to the differential expression/activity of transcriptional factors and protein kinases involved in the pathways of cellular differentiation, proliferation and tumor promotion activity. This activity may result from the direct inhibition of the protein phosphatases PP1 and PP2A. This review aims to summarize the increasing data regarding the molecular mechanisms of MC toxicity in animal systems, reporting for direct MC interacting proteins and key enzymes in the process of toxicity biotransformation/excretion of these cyclic peptides.

Dynamics of microcystins in the mussel Mytilus galloprovincialis.

The accumulation and depuration of hepatotoxins produced by the freshwater cyanobacterium Microcystis aeruginosa in the mussel Mytilus galloprovincialis was studied. Mussels were fed daily 10(5) cells/ml of the toxic cyanobacterium that produces microcystin-LR (MCYST-LR), for four days. After that period animals were placed in toxin free water and were fed the diatom Nitzschia sp. During two weeks the concentration of the toxin in the mussels, as also in their feces and in the water where animals were placed individually during 24 h, were monitored using an ELISA assay. No mussel mortality was registered during the whole experiment. Mussels showed a maximum detectable level of MCYST of 10.7 microg/g mussels dry weight (DW) during the accumulation period, rising to 16.0 microg MCYST/g mussel DW by day two of the depuration period. Then there was a decrease trend with peaks of toxin at days 6, 8, 11 and 14. The rise of the toxin level on day two of the depuration period seems to have been due to the reingestion of contaminated feces. In fact, feces showed high amounts of MCYST during the first days of depuration with a maximum of 140 microg/d DW on day 3. This coincided with a 50% decrease on the detectable toxin in the mussels reflecting the emptiness of their digestive tract. In the water the highest level of the toxin was 2.5 microg MCYST/liter and some toxin peaks were also observed during the depuration period. This fluctuation of the toxin levels in the mussels, feces and water may be related to the renewal of protein phosphatases and subsequent release of unbound toxins. Results show that depuration of MCYST by mussels is not a very rapid process and contamination by feces containing MCYST is likely to occur and increase the persistence of these toxins in the mussels after the bloom disappearance. Monitoring programs for harmful algal blooms usually include only toxic dinoflagellates and diatoms and their toxins in bivalves. Taken into account the present work they should also include hepatotoxins from cyanobacteria, namely in brackish waters such as estuaries of eutrophic rivers in order to avoid human health hazard.

Uptake and depuration of the heptapeptide toxin microcystin-LR in Mytilus galloprovincialis

Mussels (Mytilus galloprovincialis Lamarck) were exposed to the toxic cyanobacterium Microcystis aeruginosa Kutz. emend Elkin for 16 days in order to study bioaccumulation of the cyclic heptapeptide toxin microcystin-LR. After this time period the animals were fed marine phytoplankton for 2 weeks and the amount of microcystin in the mussels was monitored again. During the exposure phase mussels attained a maximum of 10.5 μg of toxin per g dry mussel weight at day 10. Microcystin was detectable after the second day of exposure. The percentage of toxin that was taken up relative to the total amount administered varied from 24.1% to 54.8%. When the mussels were fed marine phytoplankton there was a 50% decrease in the amount of detectable toxin in the mussels within 2 days. This was followed by a slight increase in toxin levels over 5 days with no microcystin-LR being detected at day 13. The major part of the toxin (96%) was found in the digestive gland + stomach while the gills, muscle, foot, and other organs combined had less than 4% of the total toxin. The results presented here show that cyanobacteria blooms present in estuaries where bivalves are growing naturally constitute a health hazard in that these organisms can retain microcystins and transfer them through the food chain.

First report and toxicological assessment of the cyanobacterium Cylindrospermopsis raciborskii from Portuguese freshwaters

The freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in freshwaters worldwide. This species is a concern from a water quality perspective due to its known ability to produce a potent hepatotoxic alkaloid cylindrospermopsin, which has been implicated in outbreaks of human sickness and cattle mortality. C. raciborskii strains isolated from Brazil have also been found to produce the highly toxic paralytic shellfish poisons (PSPs). This article reports the toxicity of four strains of C. raciborskii taken from three reservoirs and one river in Portugal, as well as the occurrence of this species in other water bodies used for potable and recreational purposes. All four strains grown in pure culture in the laboratory were found to be toxic in the mouse bioassay at 8-24h after intraperitoneal administration of single doses ranging from 1337 to 1572 mgkg(-1) Histological examination indicated that liver damage was the primary lesion; in addition, there was inflammation in the intestine. HPLC/MS tests for the presence of cylindrospermopsin, microcystins, and PSP toxins were negative. The available evidence suggests that another toxin may be present. This constitutes the first report of toxic C. raciborskii in Europe and draws attention to the need for increased monitoring of this cyanobacterium in water bodies used for potable and recreational purposes.

Hepatotoxic microcystin diversity in cyanobacterial blooms collected in portuguese freshwaters

Abstract Twelve toxic cyanobacterial bloom samples collected in natural lakes, reservoirs and rivers of Portugal were analysed. Toxicity was evaluated by mouse LD 50 bioassay of the lyophilised samples. The main bloom species present in the samples were Microcystis aeruginosa, Microcystis wesenbergii, Anabaena flos-aquae and Nostoc sp. Toxins were extracted, isolated by reverse phase HPLC and characterised by HPLC amino acid analysis and fast atom bombardment mass spectrometry. Two to seven microcystins were purified from each sample, and a total of seven different toxins were isolated and their structure assigned. MCYST-LR was the most common and its proportion in each sample ranged from 45.5% to 99.8% of the total microcystin contents. MCYST-RR, MCYST-YR and [D-Asp 3 ]MCYST-LR were also identified in the samples. Three less common microcystins, MCYST-HilR, [L-MeSer 7 ]MCYST-LR and [Dha 7 ]MCYST-LR, were found in only one sample. Total MCYST concentration varied from 1.0 to 7.1 μg/mg dry weight of cyanobacteria. Significant relationships between LD 50 -MCYST-LR-total MCYST content were found. The need for monitoring cyanobacteria and their toxins in eutrophic waters that are used for drinking and recreation purposes is discussed.

Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview

The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na+ and K+ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins.

Cyanobacteria diversity and toxicity in a Wastewater Treatment Plant (Portugal)

Cyanobacteria are common in eutrophic natural waters. Being favoured by warm, stable and nutrient-enriched waters they may constitute an important part of the phytoplankton community in Wastewater Treatment Plants (WWTP). The phytoplankton communities of two ponds (facultative and maturation) of the WWTP of Esmoriz (North Portugal) were studied, with particular importance given to cyanobacteria. Mouse bioassays were performed with cyanobacteria samples during some of the blooms and ELISA assays specific for hepatotoxic microcystins were carried out. During the study period (January-July 1999) cyanobacteria were frequently dominant in the ponds ranging from 15.2 to 99.8% of the total phytoplankton density. The main species were Planktothrix mougeotii, Microcystis aeruginosa and Pseudanabaena mucicola. Mouse bioassays were performed during Oscillatoria bloom period but the results were negative, in spite of the high cyanobacteria biomass. ELISA assays were performed for both ponds but only in the maturation pond positive values were found. Microcystin concentrations (as MCYST-LR equivalents) varied from 2.3 to 56.0 micrograms/l on the margin of the pond and between 1.7 and 4.6 micrograms/l in the outflow of this pond. These values indicate that WWTP may be a source of contamination of water bodies with cyanobacteria toxins.

Isolation, characterization and quantification of microcystins (heptapeptides hepatotoxins) in Microcystis aeruginosa dominated bloom of Lalla Takerkoust lake–reservoir (Morocco)

This paper presents the first data on the identification, characterization and quantification of microcystins isolated from both an extract of a cyanobacteria natural bloom, collected from a eutrophic Moroccan reservoir (Lalla Takerkoust, Marrakesh) and an isolated strain cultivated under laboratory conditions. The isolation and purification of toxins was performed by reverse phase HPLC and then characterized by amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS). Chemical characterization of the toxins from the bloom revealed variants of microcystins such as Mcyst-LR, Mcyst-RR, Mcyst-YR and [D-Asp3]Mcyst-LR. However, the Microcystis aeruginosa strain produced only Mcyst-RR. Using an ELISA assay the total microcystin contents of eight bloom samples collected from 1994 to 1997 ranged from 0.7 to 8.8 microg/mg of lyophilized material. The two isolated Microcystis strains contained higher amounts of microcystins (0.65 microg/ mg of dry weight) than the Pseudanabaena strains (0.021 microg/mg of dry weight). Our results show that the presence of cyanobacteria toxins in water used for drinking in a North African country may be regarded as an health hazard. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, namely in north African countries.

Impact of a toxic and a non-toxic strain of Microcystis aeruginosa on the crayfish Procambarus clarkii

The occurrence of cyanobacteria in many water bodies where crayfish such as Procambarus clarkii are abundant leads to the possibility of toxin accumulation and food chain transfer. This paper describes the accumulation and depuration of microcystins from a microcystin and a non-microcystin producing strain of Microcystis aeruginosa, on the survivorship, growth and nutritional status of P. clarkii. Crayfish larvae were resistant to cyanobacteria and their toxins, surviving cyanobacteria densities during acute exposures. Juvenile crayfish tolerated toxic cyanobacteria better than non-toxic ones. This effect was also observed when analysing nutritional status of crayfish fed toxic and non-toxic cyanobacteria with the former having better lipid and protein contents than those fed non-toxic Microcystis. P. clarkii accumulated up to 2.9 microg MCYST/dry crayfish weight and the depuration pattern was similar to that observed for mussels by other authors. Due to the fact that the major part of the toxin is accumulated in the intestine and in the hepatopancreas, there is no significant risk in terms of human health if these parts are removed prior to crayfish consumption. Nevertheless, their use in dairy food and the possible transference of toxins along food chains should not be disregarded.

Allelopathy in freshwater cyanobacteria.

Freshwater cyanobacteria produce several bioactive secondary metabolites with diverse chemical structure, which may achieve high concentrations in the aquatic medium when cyanobacterial blooms occur. Some of the compounds released by cyanobacteria have allelopathic properties, influencing the biological processes of other phytoplankton or aquatic plants. These kinds of interactions are more easily detectable under laboratory studies; however their ecological relevance is often debated. Recent research has discovered new allelopathic properties in some cyanobacteria species, new allelochemicals and elucidated some of the allelopathic mechanisms. Ecosystem-level approaches have shed some light on the factors that influence allelopathic interactions, as well as how cyanobacteria may be able to modulate their surrounding environment by means of allelochemical release. Nevertheless, the role of allelopathy in cyanobacteria ecology is still not well understood, and its clarification should benefit from carefully designed field studies, chemical characterization of allelochemicals and new methodological approaches at the “omics” level.