Ian R. Falconer

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.