Biosynthesis of cylindrospermopsin.

Studies on the biosynthesis of cylindrospermopsin (1), a potent hepatotoxin associated with the cyanobacterium Cylindrospermopsis raciborskii, indicate that 1 is an acetogenin with guanidinoacetic acid serving as the starter unit of the polyketide chain. Feeding experiments show that C14 and C15 of 1 are derived from C1 and C2 of glycine, respectively, and C4 through C13 arise from five contiguous acetate units attached head to tail. The methyl carbon on C13 originates from the C(1) pool. The starter unit, established by the incorporation of [guanidino-(13)C,alpha-(15)N]-guanidinoacetic acid into N16 and C17 of 1, does not appear to be formed from glycine by known amidination pathways. The origin of the NH-CO-NH segment in the uracil ring is also unknown.

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function.

Inhibition of serine/threonine protein phosphatases in rat hepatocytes by okadaic acid and microcystin increased the phosphorylation of several components of the cytoplasmic dynein complex. UV light/vanadate cleavage and Western blot analysis revealed that two of these components with molecular masses of approx. 400 kDa and 74 kDa were dynein heavy- and intermediate-chains respectively. This increased phosphorylation resulted in inhibition of dynein ATPase activity, and reduced motor-dependent avidity of endosomal/lysosomal membranes for microtubules.

Prolonged sublethal exposure to the protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects.

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.

Differential toxicity of the protein phosphatase inhibitors microcystin and calyculin A.

Microcystin (Mcyst) and calyculin A (CalA) in vitro inhibit protein phosphatases (PP)1 and 2A activity (IC50 0.1-2.0 nM). This study was aimed at determining the contribution of PP inhibition to Mcyst hepatotoxicity by comparing the effect of these two chemically different inhibitors in perfused rat livers. Both compounds (60 micrograms Mcyst and 6 micrograms CalA/150 ml perfusate) caused cessation of bile flow and inhibition of PP activity after 20 min of perfusion to 8% and 37% of control activity for Mcyst and CalA treatments, respectively. Histopathological findings included loss of cord sinusoidal pattern and of normal liver architecture. There also was hepatocyte swelling, pyknotic changes and necrosis. Mcyst caused a modest increase in perfusion pressure of 1.2 cm of water, whereas CalA caused a 3-fold increase. The most likely explanation for this hemodynamic effect is direct action of CalA on the vascular endothelium and/or sinusoidal and perisinusoidal cells. This possibility was explored with hepatocytes and sinusoidal endothelial cells. PP activity of both cell types was inhibited by 10 to 100 nM CalA followed later by cell lysis, whereas Mcyst (500 nM-2 microM) had no effect on sinusoidal endothelial cells, but inhibited PP activity and caused later lysis in hepatocytes (Mcyst 20-160 nM). Mcyst hepatotoxicity is therefore a direct consequence of PP inhibition in hepatocytes, the loss of sinusoidal integrity following from the primary toxic insult to the hepatocyte. Inhibition of PP activity of the cells of the presinusoidal vasculature and/or nonparenchymal cells results in hepatic hypertension.

Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes.

Cylindrospermopsin (CY) is a naturally occurring alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in humans. We previously showed that CY is cytotoxic to primary cultures of rat hepatocytes and that CY lowers cell reduced glutathione (GSH) at nontoxic doses. Lower cell GSH also potentiates CY-induced cytotoxicity (Runnegar et al., Biochem Biophys Res Commun 201: 235-241, 1994). Our current work examined the mechanism of the fall in cell GSH induced by CY. We excluded several possible explanations for the loss in GSH, namely increased formation of oxidized glutathione (GSSG), increased GSH efflux, hidden forms of GSH, decreased GSH precursor availability, or decreased cellular ATP level. To address whether the fall in GSH was due to decreased GSH synthesis or increased GSH consumption, we examined the rate of fall in total GSH after 5 mM buthionine sulfoximine (BSO, an irreversible inhibitor of GSH synthesis) treatment. The rates of fall in total GSH (nmol/10(6) cells/hr) were 8.2 +/- 2.5, 6.0 +/- 1.7 and 5.9 +/- 1.3 for control, 2.5 microM and 5 microM CY-pretreated cells, respectively. This suggests that the fall in GSH induced by CY was due to the inhibition of GSH synthesis rather than increased consumption, because in the latter case the rate of fall in GSH would have been accelerated by CY pretreatment. Furthermore, excess GSH precursor (20 mM N-acetylcysteine), which supported GSH synthesis in control cells, did not prevent the fall in GSH or toxicity induced by CY. Treatment of cells with the cytochrome P450 inhibitor alpha-naphthoflavone protected partially from CY-mediated toxicity and from the fall in cell GSH. Thus, it is likely that cytochrome P450 is involved in the metabolism of CY, and the metabolite(s) that is generated may be more toxic and/or potent in inhibiting GSH synthesis. Inhibition of GSH synthesis is most likely an important factor in the cytotoxicity of CY.

The role of glutathione in the toxicity of a novel cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes.

Cylindrospermopsin (CY) is a newly isolated alkaloid produced by the cyanobacterium Cylindrospermopsis raciborskii, which has been linked to an outbreak of hepatoenteritis in man. The current work examined the suitability of primary cultures of rat hepatocytes as an in vitro model for studying the cytotoxicity of CY. We found that CY (3.3-5.0 microM) caused significant cell death (40-67% of cells by LDH release) in cultured hepatocytes after 18 hr incubation. While investigating possible mechanisms for CY toxicity, we found that lower, nontoxic doses of CY (1.6-2.5 microM) decreased cell glutathione (GSH) to about 50% of control. For toxic doses (5 microM), the loss of GSH preceded the onset of toxicity by six hr. Lowering cell GSH predisposed cells to CY toxicity. In conclusion, cultured hepatocytes are a suitable model for studies of CY cytotoxicity and GSH is involved in the detoxification of CY.

Protein phosphatase inhibition and in vivo hepatotoxicity of microcystins.

Administration of microcystin (MCYST)-YM or -LR (peptide hepatotoxins produced by the cyanobacterium Microcystis aeruginosa) to mice resulted in the inhibition of liver protein phosphatase 1 and 2A activity. In all cases significant inhibition preceded or accompanied clinical changes due to MCYST intoxication. Fifteen minutes after intraperitoneal injection of lethal doses of MCYST-YM protein phosphatase activity was already decreased to 44% of controls, and by 60 min was further decreased to 22% of controls. The inhibition was dose dependent: intraperitoneal injection with 84 nmol/kg of MCYST-YM and 48 nmol/kg of MCYST-LR were the minimum doses required for significant inhibition at 60 min. Pretreatment of mice with 200 mumol/kg of rifamycin prevented the inhibition of liver protein phosphatase. The inhibition was tissue specific, with none detected in the kidneys, an organ that, unlike the liver, does not accumulate MCYST. In contrast to MCYST intoxication, lethal doses of phalloidin, a peptide hepatotoxin that produces clinical and pathological changes similar to MCYST, did not cause any inhibition of protein phosphatases.

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 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.

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.

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.

Effect of toxin from the cyanobacterium Microcystis aeruginosa on ultrastructural morphology and actin polymerization in isolated hepatocytes.

Freshly isolated rat hepatocytes incubated with the hepatotoxin from the cyanobacterium Microcystis aeruginosa are rapidly deformed (blebbed). Transmission electron microscopy shows the appearance of unusual intracellular structures and rearrangement of cellular organelles, without any change in the polymerization state of actin. Cytochalasin E (20 microM), a fungal metabolite that causes blebbing of hepatocytes, had no significant effect on the polymerization state of cellular actin, but if Microcystis toxin (10 microM) was added together with cytochalasin E (20 microM), there was a significant increase (from 30% to 44%) in the proportion of unpolymerized G-actin in hepatocytes. These findings are in contrast to the effect of phalloidin (12.5 - 37.5 microM), a peptide hepatotoxin from the poisonous mushroom Amanita phalloides, which also causes blebbing of hepatocytes, and was shown in this study to decrease the level of unpolymerized G-actin in the cells to below measurable levels when added by itself or together with Microcystis toxin or cytochalasin E.

Biological half-life, organ distribution and excretion of 125-I-labelled toxic peptide from the blue-green alga Microcystis aeruginosa.

M. aeruginosa is a bloom-forming cyanobacterium which is common in fresh-water lakes. It contains a potent hepatotoxin which when purified has been shown to be a heptapeptide of molecular weight 1019. The toxin was iodinated with 125I using the lactoperoxidase method, the labelled toxin administered intravenously to adult female rats and the half-life and organ distribution measured. The blood half-life after redistribution into extracellular pools was 42 min. The liver and kidneys showed accumulation of 21.7 +/- 1.1 and 5.6 +/- 0.2% of the dose respectively after 30 min. Little accumulation was observed in other organs and tissues. Small-intestinal contents and urine contained 9.4 +/- 6.1 and 2.9 +/- 1.2% of the dose respectively after 120 min. It was concluded that the liver is the main target organ for both accumulation and excretion of the toxin.

Experimental acute intoxication of young layer and broiler chickens with the cyanobacterium (blue-green alga) Microcystis aeruginosa.

An extract of a natural bloom of the blue green alga Microcystis aeruginosa was administered to groups of 7-day-old layer and broiler commercial chickens as a single oral dose. The LD50 values ranged from 1295 to 1643 mg of freeze-dried algal bloom per Kg of chicken. The toxin was principally hepatotoxic causing massive hepatocellular necrosis and biliary hyperplasia in lethally affected chickens. Lymphoid necrosis was seen in the bursa and spleen. The intraperitoneal LD50 of the bloom in chickens and mice was determined to be 13 and 19 mg of freeze-dried bloom per Kg of chicken and mice respectively.

Lethal potency and tissue distribution of 125I-labelled toxic peptides from the blue-green alga Microcystis aeruginosa.

Toxic heptapeptides from a water bloom of the cyanobacterium Microcystis aeruginosa were purified by HPLC. The unoxidised fraction was iodinated with 125I plus 127I by the lactoperoxidase/H2O2 method, further purified by HPLC, and the non-iodinated and three iodinated fractions administered i.p. to male mice. All iodinated fractions were toxic, with symptoms and pathological lesions of the liver identical with those caused by non-iodinated peptide. Radioactivity was concentrated in the liver of mice at death.

Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir.

Cylindrospermopsis raciborskii, a tropical blooming species of cyanobacterium (blue-green alga), was isolated from the domestic water supply reservoir on Palm Island, a continental island off the tropical northeast coast of Australia. This species, not previously known to be toxic, was shown to be severely hepatotoxic for mice. The 50% lethal dose at 24 h after injection was found to be 64 +/- 5 mg of freeze-dried culture per kg of mouse. The principal lesion produced was centrilobular to massive hepatocyte necrosis, but various degrees of injury were also seen in the kidneys, adrenal glands, lungs, and intestine. The possible implication of this finding in relation to an incident of hepatoenteritis in humans living on the island is discussed.

Clinical and pathological changes in sheep experimentally poisoned by the blue-green alga Microcystis aeruginosa.

Fifteen young sheep were inoculated intraruminally with various doses of an algal bloom of Microcystis aeruginosa. Lethally poisoned sheep died between 18 and 48 hours after inoculation. Findings included marked elevation of serum concentrations of certain enzymes and bilirubin, mild elevations of blood urea nitrogen and serum inorganic phosphorus with marked reduction in blood glucose, a mild neutrophilia with a marked left shift and marked changes in coagulation parameters. Necropsy findings included pale swollen, sometimes hemorrhagic, livers, edema involving the body cavities and widespread small hemorrhages. The primary site of toxicity was the liver which had centrilobular to near massive hepatocyte necrosis. Electron microscopic lesions were aggregation of endoplasmic reticulum with displacement of subcellular organelles towards the periphery of the hepatocyte, and vacuolation of the contents of more severely affected cells.

Evidence of liver damage by toxin from a bloom of the blue-green alga, Microcystis aeruginosa.

Examination of the results of routine assays for hepatic enzymes in plasma has shown a significant elevation of the levels of gamma-glutamyl-transpeptidase, and a lesser elevation of those of alanine aminotransferase, in plasma specimens from a population who obtained drinking water from a reservoir containing a heavy bloom of toxic Microcystis aeruginosa. Such elevations did not occur in the adjacent population who did not use water from this source. They coincided with the occurrence of the algal bloom, and were not present in the corresponding assays during the periods before and after the occurrence of the bloom. Increases in the levels of these enzymes indicate toxic liver injury and could not be attributed to clinical changes in the frequency of acute liver conditions or to variations in the number of plasma samples taken from patients with alcoholism. Therefore, it is concluded that the toxic algae in the water-supply reservoir cause the observed elevation of the levels of hepatic enzymes in the plasma of the consuming population.