Investigation of the mechanisms involved in cylindrospermopsin toxicity : hepatocyte culture and reticulocyte lysate studies / Suzanne M. Froscio.

Copy of author's previously published work inserted.Bibliography: leaves 121-139.xvii, 139 leaves : ill. (some col.) ; 30 cm.The aim of this study was to determine the extent to which protein synthesis inhibition, lowered glutathione (GSH) levels and toxin metabolism contribute to the toxicity of cyclindrospermopsin. Both hepatocyte cultures and reticulocyte lysates were utilized as in vitro tools of investigation. The findings imply that the inhibition of protein synthesis by direct action of the toxin cannot be considered a primary cause of hepatocyte cell death over an acute time frame. Cytochrome P450-derived metabolites may play a crucial role in cytotoxicity, and the toxicity process does not appear to involve oxidative damage.Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2002

Novel toxic activity associated with the cyanobacteria Limnothrix : use of screening assays for detection

Development of biochemical screening assays for cyanobacterial toxins allows identification of toxins based on known activity. While this may be diagnostic for the toxin or toxin class that the assay has been designed for (e.g. microcystins, cylindrospermopsins) there is also potential to pick up new, unidentified toxins. In this example, screening cyanobacterial samples for the toxin cylindrospermopsin led to the identification of novel toxic activity from the cyanobacterium Limnothrix.The cell-free protein synthesis assay was inhibited by the Limnothrix extract as would be expected when cylindrospermopsin is present. However analysis by immunoassay (ELISA) and analytical techniques (HPLC, LC-MS) could not detect cylindrospermopsin itself. PCR amplification of genes associated with cylindrospermopsin production was also absent. Further characterisation revealed that the toxic responses induced by the Limnothrix extract could not be attributed to other known cyanobacterial toxins. Distinctive toxic effects of the Limnothrix extract in mammalian cells include significant ATP depletion and markedgranulation of cells as observed by both microscopy and flow cytometry. Current work aimsto identify and characterise the toxic agent present. This case highlights how biochemical screening assays can complement analytical techniques for the identification of toxic activity in cyanobacterial samples. Adequate validation of such techniques is required

Novel toxicity associated with a Limnothrix-like species : mouse bioassay results

Background & Aims: A search for novel cylindrospermopsin (CYN) producers found a Limnothrix strain that inhibited protein synthesis but did not contain detectable CYN or analogues (HPLC, LC-MS, ELISA), nor the CYN gene (Bernard et al 2010). Methods: Mouse bioassays (time-course up to seven days, and set times up to 24h) were performed followed by detailed post-mortem (PM) dissections and histopathology. By 3h, these reduced but clear mucoid diarrhoea (containing sloughed endothelium and blood cells) was observed. Body weights were reduced by 24h and remained below controls for 7d. Liver and spleen weights (% body weight) increased by 4h and remained high until at least 24h. Histopathology (10-24h) indicated cell death in the small intestine (widespread), liver and kidneys. White blood cell numbers increased in the circulation, spleen and lungs. Conclusions: Limnothrix produces a potent in vivo cytotoxin. It is water-extractable and produced by a commonly occurring genus. Hence it is a potential risk to drinking water sources. We are currently (1) clarifying the genetic identity of the toxic strain, (2) determining its Australian distribution, (3) purifying and characterising the active compound, and (4) investigating its mechanism of action post-mortem (PM) dissections and histopathology. Results: By 0.5h post-injection, signs included extreme sensitivity to touch and sudden noises, and hind limb weakness

Taxonomy and ecology of toxin producing Limnothrix

A new toxin produced by a Limnothrix was discovered while screening cyanobacterial samples for the presence of known and unknown toxins as part of an Australian Coal Association Research Project. It is the first time that a toxin-producing freshwater Limnothrix has been described. The features of this cyanobacterium in both field and pure cultures were consistent with the cyanobacterial species Geitlerinema unigranulatum as well as with the earlier Australian descriptions of Limnothrix cf. planctonica. However, genetic analyses closely group this cyanobacterium with the commonly occurring temperate species Limnothrix redekei. Limnothrix may occur as solitary planktonic trichomes, mats on the bottom or within the water column, or in balls floating on the surface. A fine mucilage, often confused with a sheath, may cover older trichomes. The long, thin trichomes may glide into coils, may flex and are not attenuated at the ends. The cylindrical cells are 1.6-2.0 μm wide and 5.0-6.5 μm long. Constriction at the cross walls is generally not observed and cross walls are often indistinct. Refractile granules are present in the cells - near the cell walls and sometimes distributed within the cells. Limnothrix often co-dominates with Cylindrospermopsis raciborskii in both temperate and tropical environments. In the Fitzroy River system, it is often found just above the level of the thermocline. However, toxin producing material has also been isolated from water sampled from major pipelines within the Central Queensland region

Cyanotoxins are not implicated in the etiology of coral black band disease outbreaks on Pelorus Island, Great Barrier Reef

Cyanobacterial toxins (i.e. microcystins) produced within the microbial mat of coral black band disease (BBD) have been implicated in disease pathogenicity. This study investigated the presence of toxins within BBD lesions and other cyanobacterial patch (CP) lesions, which, in some instances (∼19%), facilitated the onset of BBD, from an outbreak site at Pelorus Island on the inshore, central Great Barrier Reef (GBR). Cyanobacterial species that dominated the biomass of CP and BBD lesions were cultivated and identified, based on morphology and 16S rRNA gene sequences, as Blennothrix- and Oscillatoria-affiliated species, respectively, and identical to cyanobacterial sequences retrieved from previous molecular studies from this site. The presence of the cyanotoxins microcystin, cylindrospermopsin, saxitoxin, nodularin and anatoxin and their respective gene operons in field samples of CP and BBD lesions and their respective culture isolations was tested using genetic (PCR-based screenings), chemical (HPLC-UV, FTICR-MS and LC/MSn) and biochemical (PP2A) methods. Cyanotoxins and cyanotoxin synthetase genes were not detected in any of the samples. Cyanobacterial species dominant within CP and BBD lesions were phylogenetically distinct from species previously shown to produce cyanotoxins and isolated from BBD lesions. The results from this study demonstrate that cyanobacterial toxins appear to play no role in the pathogenicity of CP and BBD at this site on the GBR