Interaction of the cyanobacterial toxin cylindrospermopsin with the eukaryotic protein synthesis system.

The cyanobacterial toxin cylindrospermospin (CYN) was shown to inhibit the eukaryotic protein synthesis apparatus with similar potency in plant and mammalian cell extracts, IC(50) of 334 nM in wheat germ extract and 110 nM in reticulocyte lysate. [(14)C] CYN binding was assessed in reticulocyte lysates, following separation of ribosomes from free toxin on Sephadex G-50 size exclusion columns. While toxin binding was shown to be concentration-dependent (60-600 nM [(14)C] CYN), it did not correlate with ribosome content. The molar ratio of toxin to ribosomes was 0.02:1 at the binding plateau. Significant binding of [(14)C] CYN was identified in both the ribosome fraction (> 90% total ribosomes) and the first wash fraction eluting from the Sephadex G-50 column, despite the low ribosome content (<10% total ribosomes) in the latter. Bound [(14)C] CYN could be partially displaced by incubation with an excess of unlabelled CYN in both the ribosome and wash fractions, indicating a non-covalent linkage. Molecular weight cut-off filters identified that [(14)C] CYN was associated with high molecular weight material > 100 kDa. These results suggest that CYN's target may not be the ribosome itself, but rather one of the soluble proteins associated with the eukaryotic translation system.

The cyanobacterial toxin, cylindrospermopsin, induces fetal toxicity in the mouse after exposure late in gestation.

Cylindrospermopsin (cyn) is a cyanobacterial toxin implicated in human and wildlife poisonings. We have completed studies investigating the potential of purified cyn to induce developmental toxicity in mammals. The teratology study involved intraperitoneal injections (8.0-128 microg kg(-1)) on gestational days (GD) 8-12 with subsequent examination of term fetuses for viability, weight and morphological anomalies. Cyn was lethal to a significant portion of the dams receiving > or = 32 microg kg(-1). Surviving pregnant females were killed and fetuses removed for examination. Analysis indicates no adverse effects on litter size, fetal weight, or incidence of anomalies. Subsequently, 50 microg kg(-1) cyn was administered on GD 8-12 or 13-17. Animals were allowed to give birth and litters monitored for growth and viability. A reduction in litter size occurred in treated groups. Avg. pup wt. was only affected in the GD 13-17 group. GD 13-17 dams did not exhibit the toxicity noted in the GD 8-12 group but gave birth significantly earlier than controls. There was a significant number of dead GD 13-17 pups and incidences of blood in the gastrointestinal tract and hematomas in the tips of the tails in survivors. Pups were cross-fostered to control mothers in litters of 10. On postnatal days (PND) 5-6 there were no significant differences in weight gain or viability in GD 8-12 litters, while GD 13-17 litters had significantly reduced weight gain and viability. GD 13-17 exposed male pups still weighed significantly less than the controls after 15 months.

Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value.

The cyanobacterial toxin cylindrospermopsin (CYN) is a frequent contaminant of freshwaters throughout the world, including those that are sources of drinking water. The first cases of human poisoning attributed to this toxin occurred from a treated drinking water supply in Queensland, Australia, in 1979. The toxin causes extensive damage to the liver, kidneys, spleen, heart, and other organs. It is known to be a potent protein synthesis inhibitor, but there is mounting evidence for genotoxicity and that it metabolizes to even more toxic forms. As part of a risk assessment process leading to a guideline for a safe drinking water level for this toxin, we performed a series of experiments to determine a no-observed-adverse-effect level (NOAEL) for this toxin. In the first trial male mice were exposed to CYN-containing cyanobacterial extract in their drinking water (0-657 microg CYN kg(-1) day(-1)) for 10 weeks. In the second trial mice received purified CYN by daily gavage (0-240 microg CYN kg(-1) day(-1)) for 11 weeks. Body and organ weights were recorded; urine, serum, and hematology analyses were performed; and histopathological examination of tissues was carried out. Body weights were significantly increased at low doses (30 and 60 microg kg(-1) day(-1)) and decreased at high doses (432 and 657 microg kg(-1) day(-1)). Liver and kidney weights were significantly increased at doses of 240 microg kg(-1) day(-1) and 60 microg kg(-1) day(-1), respectively. Serum bilirubin levels were significantly increased and bile acids significantly decreased at doses of 216 microg kg day(-1) and greater. Urine total protein was significantly decreased at doses above 60 microg kg(-1) day(-1). The kidney appeared to be the more sensitive organ to this toxin. If it is assumed that increased organ weights and changes in functional capacity are responses to an underlying toxic effect, then the NOAEL based on this data is 30 microg kg(-1) day(-1), which, with standard calculations and uncertainty factors, provides a proposed guideline safety value of 1 microg/L in drinking water.

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin.

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.

Preliminary evidence for in vivo tumour initiation by oral administration of extracts of the blue-green alga cylindrospermopsis raciborskii containing the toxin cylindrospermopsin.

New reports indicate that the toxic alkaloid cylindrospermopsin occurs in cyanobacteria in Israel, Florida, South America, and Australia in drinking water sources. This toxin is now recognised as a potential threat to human health. Furthermore, we have recently demonstrated the mutagenicity of cylindrospermopsin in vitro in a human lymphoblastoid cell-line. Therefore it is essential to determine whether cylindrospermopsin is also carcinogenic in vivo. In this preliminary study, 53 mice were treated up to three times orally with Cylindrospermopsis raciborskii extract containing cylindrospermopsin, while 27 control mice were treated with saline. A proportion of each group were then given O-tetradecanoylphorbol 13-acetate (10 microg/mouse, twice weekly in liquid food) for the duration of the experiment; the remainder were given a control diet. After 30 weeks, the mice were euthanased and the major organs were examined histologically. Five tumours were found in 53 cylindrospermopsin-treated mice while none were found in the 27 controls. Although the number of animals used was too low to provide statistical significance (p=0.16), the calculated relative risk (RR=6.2; 95% CI: 0.33-117) indicates a potential biological and public health significance requiring further investigation. Estimates are given of the size of experiment required to provide statistical proof of cylindrospermopsin carcinogenicity.

Preliminary evidence of toxicity associated with the benthic cyanobacterium Phormidium in South Australia.

In April 2000, the water supply for Yorke Peninsula in South Australia was deemed non-potable when extracts from a proliferation of the benthic cyanobacterium Phormidium aff. formosum in Upper Paskeville Reservoir were found to be lethally toxic by intraperitoneal injection into mice (400 mg kg-1). Routine water quality monitoring had failed to detect the development of the Phormidium until complaints of musty taste and odour, attributable to the production of 2-methyl-isoborneol (MIB), were received from the consumers. The 185 ML open-balancing storage, receiving filtered and chloraminated water from the River Murray, was isolated from the drinking water supply and a health alert was issued to approximately 15,000 consumers. The identity of the toxin(s) is thus far unknown, but clinical symptoms of toxicity in mice and chemical characteristics are distinct from the known major cyanotoxins. Preliminary characterisation of this toxin indicates that it has low solubility in water and organic solvents and is strongly associated with the particulate cellular material of the filaments. Toxicity of extracts was diminished by boiling and by treatment with chlorine, but not by chloramines. Further testing of floating cyanobacterial mats in the Torrens Lake in the city of Adelaide (Phormidium aff. formosum) and Myponga Reservoir (Phormidium aff. amoenum) in 2000/2001 was also found to be toxic by mouse bioassay. Toxicity is yet to be confirmed in monospecific cultured strains and further studies are required to identify the toxin and assess its health significance. Genetic characterisation of isolates has commenced in an attempt to classify their relatedness and to assist in the rapid identification of potentially toxic strains.

Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis produced by a number of cyanobacterial species, the most common being Cylindrospermopsis raciborskii. CYN contains a uracil moiety attached to a sulphated guanidino moiety, suggesting that it may have carcinogenic activity. This report describes the use of the WIL2-NS lymphoblastoid cell-line in the well-validated cytokinesis-block micronucleus (CBMN) assay to test this hypothesis. Centromeres (CENs) were identified in micronuclei (MNi) of binucleated cells (BNCs) by fluorescent in situ hybridisation of alpha centromeric DNA sequence repeats. The results indicate that CYN induced a significant increase in the frequency of MNi in BNCs exposed to 6 and 10microg/ml, and a significant increase in CEN-positive MNi at all concentrations of CYN tested (1, 3, 6, and 10microg/ml). However, despite this apparently greater sensitivity of WIL2-NS cells to induction of CEN-positive MNi at low CYN concentrations, at the higher concentrations the magnitude of the increase in CEN-positive MNi did not account for the greater increase in MNi in BNCs, indicating that both CEN-positive and CEN-negative MNi were induced. This suggests that CYN acts to induce cytogenetic damage via two mechanisms, one at the level of the DNA to induce strand breaks, the other at the level of kinetochore/spindle function to induce loss of whole chromosomes (aneuploidy). C. raciborskii occurs in a number of human drinking water sources worldwide and so these findings may have important public health implications.

Microcystins (cyanobacterial toxins) in drinking water enhance the growth of aberrant crypt foci in the mouse colon.

Microcystis aeruginosa produces toxic cyclic peptides called microcystins, potent hepatotoxins that have been implicated in tumor promotion in skin and liver. The model used in this investigation was the azoxymethane (AOM)-induced aberrant crypt focus (ACF) in the male C57Bl/6J mouse colon. Three intraperitoneal (i.p.) injections of 5 mg/kg AOM were administered at 7-d intervals to mice; 19 d after the last AOM injection, drinking water containing Microcystis extract was commenced and continued for a further 212 d. The content of microcystins in the drinking water was determined by mouse bioassay, high-performance liquid chromatography (HPLC), capillary eletrophoresis, and protein phosphatase inhibition. The doses employed were 0, 382, and 693 micrograms/kg bodyweight/d at the midpoint of the trial. Following postmortem examination blood cells, serum enzymes and organ pathology were investigated. A significant microcystin dose-dependent increase in the area of aberrant crypt foci was observed. There was no marked increase in the number of crypts/colon. Two overt colonic tumors (approximately 30 mm3) were seen in microcystin-treated mice, and one microscopic colonic tumor in an AOM-alone-treated mouse. This investigation provides the first evidence for the stimulation of preneoplastic colon tumor growth by microcystin.

Health risks caused by freshwater cyanobacteria in recreational waters.

Toxic cyanobacteria are increasingly being perceived as a potential health hazard, particularly in waters used for recreation. A few countries are developing regulations to protect human health from these toxins, and the World Health Organization (WHO) has published both a guideline value for one cyanotoxin in drinking water and a procedural guideline for recreational waters. This article presents an overview of the currently known cyanotoxins and of documented cases of human illnesses attributed to them. It further discusses exposure pathways and approaches to risk management. In this context, the WHO guideline for recreational waters is presented, and monitoring approaches are outlined.

Isolation and toxicity of Cylindrospermopsis raciborskii from an ornamental lake.

In Australia, the tropical/subtropical cyanobacterium Cylindrospermopsis raciborskii forms substantial blooms in some drinking water supply reservoirs, rivers and recreational water bodies during the warmer months of the year. This paper describes the isolation, culture and toxicity characterisation of Cylindrospermopsis from a water bloom in a small lake in NSW, Australia. The cyanobacterium grew as straight trichomes terminating with a characteristic heterocyst. The toxic alkaloid cylindrospermopsin was separated and identified by high-performance liquid chromatography at a concentration of 5.5 mg/g dry cells, 0.026 pg/cell. Intraperitoneal injection of sonicated cells caused liver, kidney, intestinal and lung damage, with an LD50 of 52 mg cells/kg mouse body weight at 24 hr, and 32 mg/kg at 7 days. The 24 hr mouse toxicity is not consistent with previous studies using pure cylindrospermopsin, and is suggestive of other toxic compounds in this isolate.

Effect of the cyanobacterial (blue-green algal) toxins from Microcystis aeruginosa on isolated enterocytes from the chicken small intestine.

Livestock deaths, and clinical reports of human injury, follow the consumption of toxic blue-green algae. The experiments described show that isolated intestinal enterocytes from chicks are both deformed and killed by exposure to Microcystis toxins, in a dose-dependent and time-dependent manner. The enterocytes were protected from toxicity by deoxycholate, bromosulphothalein and rifampicin. It was concluded that the gastroenteritis clinically associated with accidental Microcystis ingestion is likely to reflect enterocyte injury by Microcystis toxins, and that the therapeutic use of bile acids or transport inhibitors may be of value in treatment.

Cytoskeletal changes in hepatocytes induced by Microcystis toxins and their relation to hyperphosphorylation of cell proteins.

The heptapeptide toxins produced by the blue-green alga (cyanobacterium) Microcystis aeruginosa are selectively hepatotoxic in mammals. The characteristic post-mortem pathology of the liver is extensive lobular disruption due to sinusoidal breakdown, leakage of blood into the tissue and hepatocyte disintegration. Isolated hepatocytes incubated with toxin show severe structural deformity and surface blebbing. This paper demonstrates the effects of Microcystis toxins on the contraction and aggregation of actin microfilaments, and on the relocation and breakdown of cytokeratin intermediate filaments, in cultured hepatocytes. Earlier work did not show changes in the assembly/disassembly of actin; however, this paper demonstrates the change in cytokeratin from intermediate filaments to distributed granules in the cytoplasm of toxin-affected cells. Acrylamide gel electrophoresis of cytoskeletal fractions from hepatocytes did not show changes in total cytokeratins; however, marked changes in the immunogenicity of cytokeratins at 52 and 58 kDa were seen on toxin exposure of cells. Measurement of 32P-phosphorylation of proteins in toxin-affected cells incubated with [32P]orthophosphate showed a dramatic increase compared to control incubations. This is in agreement with research elsewhere describing phosphatase inhibition in vitro by Microcystis toxins. The data indicate that phosphorylated cytokeratin is a major component of cytoplasmic fraction phosphorylated protein after toxin exposure to hepatocytes. It is concluded that the mechanism of Microcystis toxicity to the hepatocyte is through cytoskeletal damage leading to loss of cell morphology, cell to cell adhesion and finally cellular necrosis. The underlying biochemical lesion is likely to be phosphatase inhibition causing hyperphosphorylation of a number of hepatocyte proteins, including those cytokeratins responsible for microfilament orientation and intermediate filament integrity.

The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes.

Microcystin-YM a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa was radiolabeled with 125I, and used to investigate the uptake of the toxin by freshly isolated rat hepatocytes. The uptake was temperature dependent with apparent activation energy of 18 kcal/mole (77 kJ/mole) for the initial rate of uptake. Uptake of non-toxic (10-20 nM) doses of microcystin by hepatocytes continued with time, the intracellular to extracellular distribution ratio for the toxin was 70 at 60 min for 10(6) cells/ml. Uptake of higher doses of microcystin (100 nM and more) stopped when the cells blebbed: a toxic response of hepatocytes to microcystin. Uptake of microcystin by hepatocytes was inhibited 70-80% by the addition of 10 microM sodium deoxycholate or bromsulphthlein, compounds that protect hepatocytes from the toxic effects of microcystin.

Toxicity of the cyanobacterium Nodularia spumigena Mertens.

The bloom forming cyanobacterium (blue-green alga) Nodularia spumigena produced a peptide hepatotoxin with an LD50 of 70 micrograms/kg i.p. in mice. The livers of lethally poisoned mice were haemorrhagic and enlarged, the weight doubling to about 10% of total body weight. Histologically there was centrilobular to midzonal disruption and lysis of hepatocytes resulting in haemorrhage and formation of blood lakes. Death occurred approximately 1 hr after i.p. injection. By 30 min significant increases had occurred in the plasma levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and glucose paralleling degeneration and necrosis of centrilobular hepatocytes. In vitro the toxin caused rapid dose-dependent deformation of freshly isolated rat hepatocytes, which was accompanied by the activation of phosphorylase a; 125 ng/ml of toxin being sufficient to cause these changes in 10(6) cells. This work demonstrates that, both in vivo and in vitro, Nodularia toxin shares many similarities in its action to the well characterized peptide toxins of another cyanobacterium, Microcystis aeruginosa.

Oral toxicity of a bloom of the Cyanobacterium microcystis Aeruginosa administered to mice over periods up to 1 year.

Cyanobacterial blooms in lakes have been reported causing livestock deaths and liver injury to human populations. In this study bloom material consisting of Microcystis aeruginosa was collected from a farm water storage after the death of sheep drinking from it. The cyanobacterial cells were lysed and a cell-free extract was provided to mice at a series of dilutions as their only source of drinking water. Mice of both sexes, with controls, were killed at intervals up to 1 yr of administration. Autopsies, histopathological examination, and analyses of plasma lactate dehydrogenase and alanine aminotransferase were carried out. Increased mortality was observed, particularly among males, together with chronic active liver injury and elevated alanine aminotransferase in blood. In control mice and those receiving lower concentrations of extract, hepatic amyloidosis with neutrophil infiltration, and bronchopneumonia, were seen with increasing age. The bronchopneumonia appeared earlier among mice receiving cyanobacterial extract. Four tumors were seen in 71 mice receiving a high concentration of extract for up to 1 yr, none in 150 mice receiving lower concentrations, and 2 in 73 control mice. No effects on male or female fertility, embryonic mortality, neonatal viability, or skeletal development were observed, but 7 out of 73 neonatal mice born to parents given cyanobacterial extract showed reduced brain size. No cases were seen in controls. We conclude that the major toxicity exhibited is liver injury. Further attention is needed for evaluation of carcinogenicity and embryonic damage.

Toxicity to mice and sheep of a bloom of the cyanobacterium (blue-green alga) Anabaena circinalis.

A bloom of Anabaena circinalis shown to be lethal to mice (i.p. LD50 17.0 +/- 0.6 mg/kg) was tested for lethal potency when given orally to mice and intraruminally and intraperitoneally to sheep. The lethal oral dose in mice was at least 170 times the parenteral dose. The bloom was lethal when given i.p. to sheep but lethality was not observed when given intraruminally in doses up to 1710 mg/kg, equivalent to drinking 8.5 litres of thick algal bloom, a volume far in excess of that likely to be consumed naturally. In vivo testing of lethal potency by i.p. inoculation of mice is therefore an unreliable method for judging potential oral toxicity in livestock of blooms of Anabaena.

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium, pH and membrane integrity in mammalian cells.

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD50 110 micrograms/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca2+ entry by toxin (0.1-10 micrograms/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37 degrees C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 microgram/ml. No progressive Ca2+ entry was observed on toxin addition, instead a rapid rise in intracellular Ca2+, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 micrograms/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.

Injury to hepatocytes induced by a peptide toxin from the cyanobacterium Microcystis aeruginosa.

The freshwater, bloom forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin which causes death accompanied by liver necrosis. We show here that the time and dose-dependent blebbing of isolated hepatocytes is accompanied by the activation of phosphorylase a, with no changes in cyclic AMP levels, and by glutathione (acid-soluble thiols) depletion. These results suggest that the disruption of cytoskeletal structures is accompanied by disturbances in cellular calcium homeostasis and by decreased protection against oxidative damage to the cells.