Academic literature on the topic 'Cylindrospermopsin toxicity'

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.

Cylindrospermopsin (CYN) is a widely spread cyanotoxin that can occur in fresh water and food. This research aims to investigate CYN toxicity by studying the effects of drinking 0.25 nM of CYN-contaminated water from a natural source, and of the direct application of moderate concentrations of CYN on different animal targets. The chosen structures and activities are rat mitochondria inner membrane permeability, mitochondrial ATP synthase (ATPase) and rat liver diamine oxidase (DAO) activities (EC 1.4.3.22.), the force of the contraction of an excised frog heart preparation with functional innervation, and the viability of a human intestinal epithelial cell line (HIEC-6). The oral exposure to CYN decreased the reverse (hydrolase) activity of rat liver ATPase whereas its short-term, in vitro application was without significant effect on this organelle, DAO activity, heart contractions, and their neuronal regulation. The application of CYN reduced HIEC-6 cells’ viability dose dependently. It was concluded that CYN is moderately toxic for the human intestinal epithelial cells, where the regeneration of the epithelial layer can be suppressed by CYN. This result suggests that CYN may provoke pathological changes in the human gastrointestinal tract.

Cylindrospermopsin (CYN) is a widely spread cyanotoxin that can occur in fresh water and food. This research aims to investigate CYN toxicity by studying the effects of drinking 0.25 nM of CYN-contaminated water from a natural source, and of the direct application of moderate concentrations of CYN on different animal targets. The chosen structures and activities are rat mitochondria inner membrane permeability, mitochondrial ATP synthase (ATPase) and rat liver diamine oxidase (DAO) activities (EC 1.4.3.22.), the force of the contraction of an excised frog heart preparation with functional innervation, and the viability of a human intestinal epithelial cell line (HIEC-6). The oral exposure to CYN decreased the reverse (hydrolase) activity of rat liver ATPase whereas its short-term, in vitro application was without significant effect on this organelle, DAO activity, heart contractions, and their neuronal regulation. The application of CYN reduced HIEC-6 cells’ viability dose dependently. It was concluded that CYN is moderately toxic for the human intestinal epithelial cells, where the regeneration of the epithelial layer can be suppressed by CYN. This result suggests that CYN may provoke pathological changes in the human gastrointestinal tract.

Cyanobacterial (blue-green algal) toxins are known to cause poisoning in humans, livestock and wild animals. Based on their toxic mechanisms, cyanobacterial toxins are generally categorized as neurotoxins, hepatotoxins or cytotoxins. The acute oral toxicities of these toxins vary substantially, with the saxitoxins being the most toxic having an LD50 of 60 μg/kg. By comparison, the acute oral LD50 for microcystin LR (the most toxic congener) and cylindrospermopsin are approximately 5,000 to 10,000 μg/kg and 6,000 μg/kg over 5 days, respectively. There are well known adverse health issues of cyanobacterial toxin poisonings. The most serious health consequences have occurred in Brazil with the reported deaths of people from gastrointestinal symptoms associated with exposure to microcystins and cylindrospermopsin. Increased number of symptoms has also been reported via exposure to cyanobacterial toxins through water-based recreational activities. Toxins may also be present in drinking water and thus guideline values are necessary to protect the health of the population. Guideline values are available for microcystins but not for saxitoxins, cylindrospermopsin or deoxycylindrospermopsin. Considerable research is being undertaken currently on more fully understanding the mechanisms of toxicity of cylindrospermopsin to enable relevant guidelines to be established.

ABSTRACTCylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwaterCylindrospermopsis raciborskii.C. raciborskiiis an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO2affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO2affect CYN and dCYN pool size as well as the expression of the key genes,cyrAandcyrK, involved in CYN biosynthesis in a toxicC. raciborskiistrain. For cells growing at different light intensities (10 and 100 μmol photons m−2s−1), we observed that the rate of CYN pool size production (μCYN) was coupled to the cell division rate (μc) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (QCYN) and dCYN (QdCYN) are fixed. Moreover, a lack of correlation between expression ofcyrAand total CYN cell quotas (QCYNs) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO2(1,300 ppm), we observed minor effects on QCYNand no effects on expression ofcyrAandcyrK. We conclude that the CYN pool size is constitutive and not affected by light and CO2conditions. Thus,C. raciborskiibloom toxicity is determined by the absolute abundance ofC. raciborskiicells within the water column and the relative abundance of toxic and nontoxic strains.

Global warming and human-induced eutrophication drive the occurrence of various cyanotoxins in aquatic environments. These metabolites reveal diversified mechanisms of action, encompassing cyto-, neuro-, hepato-, nephro-, and neurotoxicity, and pose a threat to aquatic biota and human health. In the present paper, we review data on the occurrence of the most studied cyanotoxins, microcystins, nodularins, cylindrospermopsin, anatoxins, and saxitoxins, in the aquatic environment, as well as their potential bioaccumulation and toxicity in fish. Microcystins are the most studied among all known cyanotoxins, although other toxic cyanobacterial metabolites are also commonly identified in aquatic environments and can reveal high toxicity in fish. Except for primary toxicity signs, cyanotoxins adversely affect the antioxidant system and anti-/pro-oxidant balance. Cyanotoxins also negatively impact the mitochondrial and endoplasmic reticulum by increasing intracellular reactive oxygen species. Furthermore, fish exposed to microcystins and cylindrospermopsin exhibit various immunomodulatory, inflammatory, and endocrine responses. Even though cyanotoxins exert a complex pressure on fish, numerous aspects are yet to be the subject of in-depth investigation. Metabolites other than microcystins should be studied more thoroughly to understand the long-term effects in fish and provide a robust background for monitoring and management actions.

Algae and some Cyanobacteria, being a core part of primary production, act as a food organism for many fishes and other aquatic organisms. But they can also be responsible for fish kill or illness. Review on Cyanobacteria effect on fish growth, survival, and recruitment is the need of the hour. The mechanisms of toxicity of cyanotoxins and their toxic metabolites in fish have been scarcely covered. The effects may be sublethal on growth, physiology, survival, recruitment, and in long run, it may have a role in the fish’s adaptive response to abiotic and other biotic stressors. Around 46 species from genera of Microcystis, Cylindrospermopsis, Synechococcus, Anabaena, Lyngbya, Oscillatoria, etc. have been shown to cause toxic effects in aquatic system. The bloom of these cyanobacteria is primarily associated with altered temperature and nutrient load in water bodies due to effluents from municipal discharge and aquaculture. Their acute or chronic toxic effects may vary depending on the species, type of toxin produced, and concentration. The various cyanotoxins are grouped as hepatotoxins like microcystin, nodularin cylindrospermopsin, neurotoxins; like anatoxins, homoanatoxins, dermatotoxins; like aplysiatoxin, debromoaplysiatoxins, lyngbyatoxins, and pyrogenic component; like lipopolysaccharides (LPS). The concentration of the specific cyanotoxin in the fish body and the water along with other factors such as the length of exposure, fish metabolic processes, water parameters like dissolved oxygen and temperature, are likely to impact cyanotoxin toxicity in freshwater fish. The impact of such toxicity may be reflected on the individual species level, ecosystem level, and even at the culture system level.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 &mug/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 &mug/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-&kappaB were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-&kappaB was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-&kappaB are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several freshwater cyanobacterial species, the most prevalent in Australian waters being Cylindrospermopsis raciborskii. The occurrence of CYN-producing cyanobacteria in drinking water sources worldwide poses a potential human health risk, with one well-documented case of human poisoning attributed to the toxin. While extensive characterisation of CYN-induced toxicity has been conducted in rodents both in vivo and in primary cell cultures, little is known about mechanisms of toxicity in human cell types. This thesis describes studies undertaken to further define the molecular mechanisms of CYN toxicity in human cells. Concentration-response relationships were determined in various cultured human cell types using standard toxicity assays. As expected, CYN caused dose-dependent decreases in the growth of three cell lines, HepG2, Caco-2 and HeLa, and one primary cell type, human dermal fibroblasts, according to tetrazolium reduction assays. CYN treatment did not disrupt cellular membranes according to the lactate dehydrogenase release assay in HepG2 or Caco-2 cells after 24, 48 or 72 h exposure, but did cause membrane disruption in fibroblasts after 72 h exposure to relatively high concentrations of the toxin. Apoptosis occurred more readily in HeLa cells than HepG2 cells or fibroblasts, with 72 h exposure to 1 µg/mL required before statistically significant rates of apoptosis occurred in the latter cell types. CYN did not appear to directly affect the structure of actin filaments or microtubules under the conditions used in the present study. The major portion of the work presented in this thesis comprises a large-scale interrogation of changes in gene expression induced by the toxin in cultured cells. To assess the effects of CYN on global gene expression, relative messenger RNA (mRNA) levels in human dermal fibroblasts and HepG2 cells after 6 h and 24 h exposure to 1 µg/mL CYN were determined using oligonucleotide microarrays representing approximately 19 000 genes. Overall, the number of transcripts significantly altered in abundance was greater in fibroblasts than in HepG2 cells. In both cell types, mRNA levels for genes related to amino acid biosynthesis, carbohydrate metabolism, and protein folding and transport were reduced after CYN treatment, while transcripts representing genes for apoptosis, RNA biosynthesis and RNA processing increased in abundance. More detailed data analyses revealed the modulation of a number of stress response pathways—genes regulated by NF-?B were induced, DNA damage response pathways were up-regulated, and a large number of genes involved in endoplasmic reticulum stress were strongly down-regulated. Genes for the synthesis and processing of mRNA, tRNA and rRNA were strongly up-regulated, indicating that CYN treatment may increase the turnover of all forms of cellular RNA. A small group of genes were differentially expressed in HepG2 cells and fibroblasts, revealing cell-specific responses to the toxin. Selected changes in transcript level were validated using real-time quantitative reverse transcriptase PCR (qRT-PCR). The modulation of stress response pathways by CYN, indicated by microarray analysis, was further investigated using other methods. The role of tumour suppressor protein p53 in CYN-mediated gene expression was confirmed by measuring the expression of known p53-regulated genes following CYN treatment of HepG2 cells and human dermal fibroblasts using qRT-PCR. Western blotting of protein extracts from CYNtreated cells showed that p53 protein accumulation occurred in HepG2 cells, providing additional evidence of the activation of the p53 pathway by CYN in this cell line. The immediate-early genes JUN and FOS were found to be induced by CYN in a concentration-dependent manner, and MYC was induced to a lesser extent. The mitogen-activated protein kinase c-Jun NH2-terminal kinase, implicated in the ribotoxic stress response initiated by damage to ribosomal RNA, appeared to become phosphorylated in HeLa cells after CYN exposure, suggesting that ribotoxic stress may occur in response to CYN in at least some cell types. The expression of a reporter gene under the control of a response element specific for NF-?B was induced at the mRNA level but inhibited at the protein level. This shows that while transcription factors such as p53 and NF-?B are apparently activated in response to the toxin, transactivation of target genes may not necessarily manifest a corresponding increase at the protein level. The current work contributes significantly to the current understanding of cylindrospermopsin toxicity in human-derived cell types, and provides further insight into putative modes of action.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Faculty of Science, Environment, Engineering and Technology
Full Text

Cyanobacteria is perhaps the phylum that profit the most from the escalating hypereutrophication of continental waters. The resulting cyanobacterial blooms may accumulate a variety of potent toxins. Cylindrospermopsin (CYN) is a cyanotoxin known for inhibiting protein synthesis, and producing oxidative stress as well as DNA damage in eukaryotic cells. Since the toxin\'s molecular mechanisms and targets are still unclear, we purified the cyanotoxin from our lab strains and employed a shotgun proteomics approach to reveal the major changes in HepG2 cells at sublethal doses of CYN (1 µM for 6, 12 and 24h). Metabolically labeled cells were stimulated and lysed after each treatment, their tryptic digests were separated by nano HPLC and analyzed by high-resolution tandem mass spectrometry (HRMS) on data dependent acquisition mode. We scanned an average of 4000 proteins in every sample throughout the three timepoints. Cholesterol biosynthesis and transport was mostly downregulated throughout the timepooints of the experiment. Downregulation of proteins related to ubiquitination (e.g. UBE2L3) and proteolysis pathways (e.g. PSMA2) was observed in the proteomics dataset, and these results were validated by western blot. Transcription, translation and cell cycle processes showed convoluted regulation dynamics involving known cell cycle regulators like PCNA. Downregulation of mitochondrial enzymes, oxidative stress and damage to the mithochondrial inner membrane was early evidenced after a 6 hrs treatment and validated using a JC-1, a mitochondrial membrane potential probe. The resulting dataset gives us a first glimpse of the protein groups affected at the early stage of CYN cell intoxication.
Cianobactéria é o filo que mais se beneficia da crescente hipereutrofização das águas continentais. As florações de cianobactérias resultantes podem acumular potentes toxinas. A Cilindrospermopsina (CYN) é uma cianotoxina conhecida por inibir a síntese protéica e produzir estresse oxidativo, além de danos ao DNA em células eucarióticas. Os mecanismos moleculares e alvos de toxicidade aguda desta toxina ainda não são claros. Por esse motivo adoptamos uma abordagem de proteômica quantitativa baseada em descoberta para revelar as principais alterações nas células HepG2 em doses subletais de CYN (1 µM para 6, 12 e 24h). As proteinas dos hepatócitos foram marcadas metabolicamente, foram estimuladas com a toxina e os digestos trípticos foram analisados por espectrometria de massa em tandem de alta resolução (HRMS) no modo de aquisição dependente de dados. Escaneamos uma média de 4000 proteínas ao longo dos intervalos de tempo. A biosintese e transporte do colesterol foi inibida durante a maior parte do tratamento com a toxina. Proteínas e enzimas relacionadas com o processo de ubiquitinação (ex. UBE2L3) e proteólise (ex. PSMA2) foram inibidas, e alterações nas proteínas envolvidas nesses processos foram validadas por meio de Western Blot. Os processos de transcrição, tradução e ciclo celular mostraram uma dinâmica de regulação complexa, envolvendo reguladores e disruptores do ciclo celular como por exemplo PCNA. Danos à membrana mitocondrial e evidência de estresse oxidativo foram detectados após 6 horas de tratamento, e essas mudanças no proteoma foram validados por meio do corante JC-1 (test que detecta mudanças no potencial da membrana mitocondrial). O banco de dados resultante nos dá um primeiro vislumbre das proteinas afetados no estágio inicial da intoxicação celular pela CYN.

Bibliography: leaves 121-139. 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.

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.

Cylindrospermopsin (CYN) is an alkaloid toxin produced by at least six bluegreen algal genera. In Australia, the most common producer organism is Cylindrospermopsis raciborskii. This research determined the toxic effects and potential for toxin bioaccumulation associated with toxin-producing C. raciborskii, using environmentally relevant exposure scenarios. Short-term semi-static renewal ecotoxicity tests were conducted on four freshwater test organisms, using freeze-thawed C. raciborskii whole cell extracts or live cultures of C. raciborskii containing CYN. Duckweed (Spirodela oligorrhiza) demonstrated variable responses to whole cellextract exposures containing 0 – 500 μg L-1 of CYN. Growth stimulation and growth inhibition were recorded from the duckweed, depending on toxin exposure concentrations and length of the incubation period. Chlorophyll content of Spirodela was also affected by toxin exposure, with small peaks in chlorophyll a recorded in conjunction with exposure to 8 μg L-1 CYN. Changes to frond morphology (chlorosis, necrosis) and reduced root lengths were also noted, but these effects were not consistent throughout all trials. Bioconcentration of free CYN was not detected in the tissues of Spirodela; small quantities of toxin recovered from the tissues probably represented toxin sorption to the cell walls. Growth of water thyme (Hydrilla verticillata) was stimulated by exposure to whole cell extracts of C. raciborskii containing a maximum of 400 μg L-1 CYN. Exposure to the test solutions appeared to promote the redistribution of plant resources in H. verticillata, and the possible benefits of this, particularly with respect to increased root production, were considered. Effects on the chlorophyll content of Hydrilla were variable and included decreases in total chlorophyll content and changes to the chlorophyll a:b ratio. Bioconcentration of free toxin was not detected in the tissues of Hydrilla. In trials with Melanoides tuberculata, exposure to whole cell extracts or live algal cultures did not significantly affect the behaviour or relative growth rates of adult snails. However, changes in the number of hatchlings released from parent snails were recorded: exposure to whole cell extracts corresponded with increased numbers of hatchlings compared with controls, whereas decreases in hatchling numbers compared with controls were recorded from treatments containing live C. raciborskii. Both bioconcentration and bioaccumulation occurred in the soft tissues of snails, although exposure to whole cell extracts resulted in only minor tissue contamination compared with that from live C. raciborskii exposures. Bioaccumulation of the analog deoxy-CYN was recorded in the soft tissues. M. tuberculata did not bioconcentrate CYN in the shell. Tadpoles of the cane toad (Bufo marinus) featured the most dramatic responses to the test solutions. Exposure to live C. raciborskii cultures resulted in up to 66% mortality of B. marinus, whereas all tadpoles survived exposure to whole cell extracts of comparable CYN concentrations. Decreases in the time spent swimming and relative growth rates were recorded from surviving tadpoles during both types of exposure regimes. Histological examination of Bufo tissues revealed tissue injuries to multiple organs, with particular severity noted in the liver, intestine, nephric ducts and gill epithelia. The extent of cellular damage was similar in whole cell extract (containing a maximum of 400 μg L-1 CYN) and the live culture trials (containing a maximum of 232 μg L-1), despite the unequal toxin concentrations. Bioconcentration of CYN was not evident during the whole cell extract trial, whereas exposure to live cultures resulted in maximum average tissue concentrations of 895 μg toxin kg-1 fresh weight. A secondary aim of the thesis was to identify and address gaps in management approaches for toxin bioaccumulation and possible environmental effects associated with toxin-producing C. raciborskii blooms. A predictive management strategy was developed to determine the likelihood of tissue contamination in aquatic organisms inhabiting water bodies affected by blooms. The ten-step framework for predicting bioaccumulation risk was based upon characteristics of cyanotoxin bioavailability, exposure and uptake routes during the progression of a toxic bloom. Key concepts included monitoring changes in toxin availability throughout the progression of a toxic bloom, and the prediction of bioaccumulation risks based on threshold toxin values for selected aquatic organisms. These threshold values were re-examined following the completion of the laboratory trials. Current approaches for the management of blooms with respect to environmental risks were discussed, and procedures for the proper evaluation of environmental risks associated with Cylindrospermopsis blooms were assessed. These included adequate detection and monitoring systems, setting of environmental guideline values, and options for the control and remediation of toxic blooms.

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?