Search

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits intracellular serine/threonine protein phosphatases causing disruption of actin microfilaments (MFs) and intermediate filaments (IFs) in hepatocytes. This study compared the effects of MCLR on the organization of MFs, IFs, and microtubules (MTs) in hepatocytes and nonhepatocyte cell lines and determined the sequence of toxin-induced changes in these cytoskeletal components. Rat renal epithelial cells and fibroblasts were incubated with MCLR at 100 or 200 microM for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 microM for 2-64 min. Cells were fixed and incubated with primary antibodies against beta-tubulin, actin, and vimentin or cytokeratin IFs, followed by gold-labeled secondary antibodies with silver enhancement of the gold probe. The fraction of fibroblasts and hepatocytes with altered cytoskeletal morphology was evaluated as a function of MCLR dose and exposure time to assess the sequence of changes in cytoskeletal components. Changes in fibroblasts and some hepatocytes were characterized initially by disorganization of IFs, followed rapidly by disorganization of MTs, with the progressive collapse of both cytoskeletal components around cell nuclei. Many hepatocytes exhibited MT changes prior to effects on IF structure. Alterations in MFs occurred later and included initial aggregation of actin under the plasma membrane, followed by condensation into rosette-like structures and eventual complete collapse into a dense perinuclear bundle. The similarity of effects among different cell types suggests a common mechanism of action, but the independent kinetics of IF and MT disruption in hepatocytes suggests that there may be at least 2 sites of phosphorylation that lead to cytoskeletal alterations.

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins from previously identified blooms of the blue-green alga Microcystis aeruginosa.

A cyanobacterial (blue-green algal) bloom containing Microcystis aeruginosa (dominant), M. viridis, and M. wesenbergii, was collected from Homer Lake (Illinois, U.S.A.) in the summer of 1988 and microcystins were isolated. One microcystin of substantially reduced toxicity was isolated, together with ten hepatotoxic microcystins. The compound with reduced toxicity was nonlethal at 1 mg/kg (i.p. mouse) and was determined to have a (C3H7O2) mono-ester of the alpha-carboxyl on the Glu unit of microcystin-LR. The other nine microcystins apart from MCLR had approximate LD50S ranging from 97 micrograms/kg to 750 micrograms/kg.

The reversibility of inhibition of plasma, red blood cell (RBC), and diaphragm cholinesterase (ChE) and clinical signs in mice given anatoxin-a(s) [antx-a(s)], a ChE inhibitor from Anabaena flos-aquae NRC-525-17, were characterized and compared with the effects of 2 known ChE inhibitors, the organophosphorus compound paraoxon and the carbamate pyridostigmine bromide. To follow recovery of ChE activity, mice were given either a control solution or an LD40 dose of one of the toxicants ip and killed at time points up to 8 d postdosing. After dosing, mice were monitored for diarrhea, fasciculations, respiratory difficulty, salivation, and tremors. In general, clinical signs in mice given antx-a(s) persisted longer than in mice given pyridostigmine and were more similar in duration to the clinical signs in mice given paraoxon. Histologic lesions were not detected in tissues of mice killed after administration of antx-a(s). Anatoxin-a(s) inhibited lesions were diaphragm ChE for greater than 1 but less than 2 d and RBC ChE for 8 d. The time required for recovery from Antx-a(s)-induced inhibition of ChE in plasma, RBC, and diaphragm was similar to or longer than that with paraoxon and longer than that with pyridostigmine. Based on the duration of antx-a(s) induced clinical signs and ChE inhibition in mice, antx-a(s) appears to be an in vivo irreversible inhibitor of ChE.

The indirectly evoked compound action potentials (ECAP) of the plantar muscles of the rat were used to investigate the pharmacodynamics in vivo of the neuromuscular blockade produced by anatoxin-a. Onset time to maximum depression and the magnitude of maximum depression in amplitude of the ECAP were dose-dependent. The mean maximum percent depression (+/- S.D.) of the ECAP induced by single, supramaximal stimulations of the posterior tibial nerve after i.v. doses of (+)anatoxin-a hydrochloride at 0, 50, 100, 200 and 800 micrograms/kg were 3 (4), 53 (15), 82 (7), 95 (2), and 100 (1), respectively. The ED50 (95% confidence limits) for depression of the ECAP was 47 mg/kg (39-57 micrograms/kg). Rats administered 200 micrograms/kg or less of (+)anatoxin-a hydrochloride had 75% return of the pretoxin amplitude of the ECAP within 93 min. Animals dosed at 800 micrograms/kg did not have return of neuromuscular function and died despite mechanical ventilation, suggesting a lethal mechanism(s) of action in addition to respiratory paralysis. Percent decrements (+/- S.D.) in the amplitude of the fourth ECAP following repetitive stimulation at 10 Hz were 6 (5), 13 (22), 46 (18) and 59 (8) from (+)anatoxin-a hydrochloride given i.v. at 0, 50, 100 and 200 micrograms/kg, respectively. The decrement observed following repetitive stimulation was attributed to a presynaptic site of action. No change in maximal motor nerve conduction velocity or latency of the ECAP was observed after i.v. administration of (+)anatoxin-a hydrochloride at 100 micrograms/kg. LD50 values (95% confidence limits) for anatoxin-a administered i.v. to mice were 386 micrograms/kg (365-408 micrograms/kg, for (+)anatoxin-a hydrochloride and 913 micrograms/kg (846-985 micrograms/kg) for racemic anatoxin-a hydrochloride. No deaths were observed in mice after i.p. administration of (-)anatoxin-a hydrochloride at doses up to 73 mg/kg.

The pathophysiologic effects of anatoxin-a(s) from the cyanobacterium Anabaena flos-aquae NRC-525-17 were investigated in anaesthetized adult male Sprague Dawley rats given the toxin by continuous intravenous infusion until death. Rats (n = 6) pretreated with atropine sulfate (50 mg/kg) intraperitoneally survived significantly longer (P less than 0.05) than non-atropinized rats (n = 6), suggesting that the muscarinic effects of anatoxin-a(s) were important in the lethal syndrome. In contrast to rats only given toxin, rats that were pretreated with atropine had a decrease in heart rate and mean blood pressure that followed profound reductions in respiratory tidal and minute volume, suggesting that neuromuscular blockade of the muscles of respiration was the cause of death. Even when survival time of rats was increased by pretreatment with atropine, phrenic nerve amplitude increased, indicating a lack of a depressive effect of anatoxin-a(s) on central mediation of respiration. Rats (n = 3) continuously ventilated during toxin infusion survived a dose more than 4 fold greater than a consistently lethal dose of the toxin. Thus, the cardiovascular effects of anatoxin-a(s) alone could not account for the death of rats. Electromyographic activity recorded from the diaphragms of rats (n = 5) during continuous toxin administration revealed an increase in muscular electrical activity that became more random and finally decreased prior to death, suggesting a toxin-induced neuromuscular blockade in vivo which ultimately was the cause of death of the anatoxin-a(s) dosed rats.

Anabaena flos-aquae produces at least two neurotoxins termed anatoxin-a (ANTX-a) and -a(s) [ANTX-a(s)]. ANTX-a is a potent postsynaptic depolarizing neuromuscular blocking agent. Its structure is the secondary amine, 2-acetyl-9-azabicyclo[4.2.1]non-2-ene. Known isolation and analysis methods have not always given satisfactory results. Therefore, an improved procedure was developed for both isolation and analysis of ANTX-a which involves four steps; extraction, clean-up, separation and determination. Extraction was efficiently done with 0.05 M acetic acid, and a reversed phase. ODS (octadecylsilanized) and a cation exchanger (COOH) organosilans bonded to silica gels were applied to a clean-up step. Separation and determination were then performed using two TLC and HPLC chromatographies.

Male Balb/C and Swiss Webster (SW) mice were administered various i.p. doses of microcystin-LR (MCLR) to establish dose-response curves and to determine if a sublethal dose of MCLR would protect against an approximate LD100 min given 2 or 3 days later. Micocystin-LR has an extremely steep dose-lethal response curve in BC mice--LD50 = 32.5 micrograms (micrograms)/kg, approximate LD0 max = 25 micrograms/kg and approximate LD100 min = 40 micrograms/kg. Liver weights increased 64% (BC) and 51% (SW) and kidney weights increased 32% (BC) and 20% (SW) within 200 minutes following administration of an approximate LD100 min of MCLR in naive mice. Grossly and histologically the marked increase in liver weight appeared to be caused primarily from intrahepatic hemorrhage and death is probably a result of hemorrhagic shock. Twenty-four hours following administration of a sublethal dose of MCLR to naive BC mice, liver weights were increased significantly (8.7%), but no clinical signs or histologic lesions were observed. In SW mice, administration of a LD23 of MCLR resulted in significantly increased survivability and survival times when an approximate LD100 min of MCLR was given 3 days later. Survivors of the LD23/LD100 min regimen had 96 hour postdosing liver weights not significantly different from those of mice which died acutely after the same hepatotoxin treatments. These survivors showed weakness, recumbency, anorexia, and icterus, and had marked gross liver lesions. Histologically these lesions were undergoing rapid reparative processes.

Adult male Long-Evans rats were injected intraperitoneally with 1.5, 3.0 or 9.0 micrograms/kg of anatoxin-a(s) that had been extracted from laboratory-grown Anabaena flos-aquae NRC-525-17, 800 micrograms/kg of paraoxon, or a control solution. Blood, anterior spinal cord, and brain cerebellar, cortical, medullary, midbrain, hippocampal, hypothalamic, olfactory and striatal cholinesterase activity was determined in rats that died prior to 2 hours or were anesthetized and killed at 2 hours. Unlike paraoxon, anatoxin-a(s) did not cause detectable inhibition of cholinesterase in the central nervous system, but did cause inhibition of cholinesterase in blood, suggesting that anatoxin-a(s) is strictly a peripheral cholinesterase inhibitor.

The effects of anatoxin-a(s) [antx-a(s)] from the cyanobacterium Anabaena flos-aquae NRC-525-17 on mean arterial blood pressure, heart rate, respiratory rate, tidal volume, minute volume, and phrenic nerve activity were evaluated in anesthetized Sprague-Dawley rats. Anatoxin-a(s) was administered by continuous intravenous infusion. The initial effect of the toxin was to slow the heart rate and reduce arterial blood pressure, followed by much more pronounced reductions in these parameters. The marked decline in heart rate and blood pressure frequently occurred before there was a large decrease in respiratory minute volume [reduced by only 15.4 +/- 3% (mean +/- S.E.) compared to the predose period], suggesting that antx-a(s) has an important muscarinic action on the cardiovascular system in vivo. Phrenic nerve amplitude increased, but, nevertheless, tidal and minute volumes decreased progressively, indicating that antx-a(s), unlike most low-molecular-weight organophosphorus cholinesterase inhibitors, does not have any remarkable inhibitory action on central mediation of respiration.

Anatoxin-a(s), an alkaloid neurotoxin from the freshwater cyanobacterium, Anabaena flos-aquae NRC-525-17, was compared to paraoxon, physostigmine and pyridostigmine for effects on brain cholinesterase after i.p. injection into Balb/c mice. The duration of clinical signs in mice injected with anatoxin-a(s) persisted longer than in mice given the carbamates and was comparable with that of paraoxon. Anatoxin-a(s) did not inhibit brain cholinesterase activity suggesting that this toxin is unable to cross the blood-brain barrier.