Stephen B. Hooser

Toxicity of Microcystin LR, a Cyclic Heptapeptide Hepatotoxin from Microcystis aeruginosa, to Rats and Mice

Abstract. Rats (Sprague-Dawley) and mice (Balb/c) were given microcystin LR intraperitoneally and were killed at intervals up to 24 hr (rats) or 90 min (mice) and necropsied. The lowest consistently lethal dose was 160 μg/kg in rats and 100 μg/kg in mice. Rats that were clinically unaffected had no lesion. All clinically affected rats in all dose groups died (from 20 to 32 hr after toxin) and had similar hepatic lesions. Livers were enlarged and dark red beginning 40 to 60 min after toxin. Mild disassociation and rounding of centrilobular hepatocytes developed within 20 min. By 60 min after toxin, degeneration and necrosis of hepatocytes involved most of the lobules except for small per portal zones. Weights of livers and kidneys were significantly increased. Eosinophilic fibrillar material filled renal glomerular capillaries as early as 9 hr after toxin. At 18 to 24 hr there was moderate vacuolation of proximal tubular epithelium with mild tubular dilatation. Beginning at 1 hr, intact hepatocytes and hepatic debris were present in pulmonary vessels. Analysis of serum revealed an increase in alanine aminotransferase 40 min after toxin; at 6 to 12 hr there were significant increases in alkaline phosphatase, total bilirubin, blood urea nitrogen, and creatinine. Mice survived only 60 to 90 min after toxin. Hepatic lesions were similar to those in rats, but renal and pulmonary lesions were not seen.

Actin Filament Alterations in Rat Hepatocytes Induced In Vivo and In Vitro by Microcystin-LR, a Hepatotoxin from the Blue-green Alga, Microcystis aeruginosa

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 μg/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 μg/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions. MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.

Algae intoxication in livestock and waterfowl.

Blue-green algae toxins include (1) hepatotoxic peptides that are known to be toxic to cattle, dogs, swine, waterfowl, and sometimes other species; (2) a nicotinic agonist neurotoxin that appears to be toxic to a wide range of animal species; (3) a peripheral-acting cholinesterase inhibitor that is very toxic to swine, birds, and dogs; (4) toxins that impair nervous transmission by blocking sodium channels in nerve cells; and (5) lipopolysaccharide endotoxins. This article provides current information on the mechanisms of action of the primary toxins recognized to date as well as on procedures important in the diagnosis and management of some of the more common cyanobacterial toxicoses in livestock and waterfowl.

Diagnostic and Clinically Important Aspects of Cyanobacterial (Blue-Green Algae) Toxicoses

Previous efforts have been made to provide concise summaries on the hazards of toxicoses from exposures of domestic animals to blue-green algae. It is clear, however, that veterinarians need improved access to information currently emerging with regard to blue-green algae toxicoses. Recent investigations are shedding light on the identity of the potent toxins responsible and the pathophysiology of the syndromes produced. Reviews from the past few years provide an idea of the reported occurrence of cyanobacterial toxicoses and the toxins detected, but the diagnosis of blue-green algae toxicosis has remained difficult because of a lack of concise information on appropriate diagnostic procedures. One must recognize that many algal blooms are not hazardous; therefore, a diagnosis of toxicosis following ingestion of an algal bloom, even when it is dominated by organisms known to have produced toxins in the past, should be confirmed. At the present time, demonstration of toxins in the algae and documentation of appropriate responses in the animal form the basis for many diagnoses.

Microcystin-LR-induced ultrastructural changes in rats.

The ultrastructure of hepatic, pulmonary, and renal lesions was evaluated in rats injected intraperitoneally with a lethal dose of microcystin-LR (MCLR, 160 μg/kg), a cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Hepatic lesions were first Seen at 10 minutes post-dosing and consisted of mild widening of hepatocyte intercellular spaces centrilobularly. At 20 minutes post-dosing, hepatocyte plasma membrane alterations were more pronounced, consisting of plasma membrane invagination with formation of variably sized and shaped intracytoplasmic vacuoles, loss of microvilli along the sinusoidal face, and widespread, pronounced hepatocyte separation. By 30 minutes, the space of Disse was markedly widened. At 60 minutes post-dosing, centrilobular areas contained necrotic cells and apparently intact, isolated, organelles intermingled with erythrocytes and platelets. In less severely affected regions there was prominent hepatocyte rounding, and erythrocytes and platelets were present in the widened space of Disse. Large amounts of hepatocellular debris and intact hepatocytes were present in the pulmonary vasculature, while smaller amounts of debris were also seen in the glomerular and peritubular capillaries of the renal cortex. This study shows that initial lesions are confined to Shape changes in the plasma membrane of hepatocytes. These changes are consistent with the hypothesis that microcystin-LR induces alterations in the hepatocyte cytoskeleton. Later changes consist of hepatocyte disassociation and necrosis, as well as endothelial damage, which allow release of hepatocytes and debris into the circulation with microembolism in lungs and kidneys.

Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver.

Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ( [3H]-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Degenerative myelopathy and vitamin A deficiency in a young black-maned lion (Panthera leo).

Degenerative myelopathy and vitamin A deficiency were diagnosed in a 1-year-old, female, black-maned lion (Panthera leo). Diffuse white matter degeneration characterized by dilated myelin sheaths, Wallerian degeneration, and reactive astrocytosis was present at all levels of the spinal cord. With luxol fast blue–cresyl echt violet stain, bilaterally symmetrical demyelination was observed in the fasciculus cuneatus of the cervical spinal cord and in peripheral white matter of cervical, thoracic, and lumbar segments. Additionally, the ventral gray columns and brain stem nuclei contained rare chromatolytic neurons with abnormal neurofilament accumulation. Leptomeninges of the cervical spinal cord were focally adhered to the dura and thickened by fibrosis and osseous metaplasia. Vitamin A deficiency was diagnosed based on hepatic vitamin A concentration of 1.71 μg/g dry weight. Adequate hepatic vitamin A concentration for yearling to adult domestic animals ranges between 150 and 1,000 μg/g dry weight. Lesions were distinct from those previously described in young captive lions with vitamin A deficiency, which had thickened skull bones and cerebellar herniation. The pathogenesis of vitamin A–associated myelopathy in this lion may be similar to that described in adult cattle, which is believed to result from spinal cord compression secondary to elevated pressure of cerebrospinal fluid.

Toxicity of Yew (Taxus spp.) Alkaloids

Yew plant intoxication has been implicated in numerous animal and human poisoning cases. The poisonous nature of the yew plant ( Taxus spp.) is attributed to the presence of cardiotoxic taxine alkaloids. The mechanism of action of taxine alkaloids involves calcium channel antagonism in cardiac myocytes. Ultimately, these toxins cause cardiac dysrhythmias and rapid onset of adverse clinical signs, often ending in death. Diagnosis of yew poisoning is frequently based on history of exposure or identification of yew plant fragments in the digestive tract; however, improvements in analytical toxicology have made chemical confirmation of the taxine alkaloids in diagnostic cases possible. In suspect cases of yew intoxication, the primary treatment for exposed mammals primarily involves symptomatic and supportive care.

Effects of storage conditions and hemolysis on vitamin E concentrations in porcine serum and liver.

Vitamin E (α-tocopherol) is an antioxidant vitamin important in protecting unsaturated fatty acids in lipid membranes from peroxidation. Variation in collection, storage, and shipping conditions of samples can potentially lead to breakdown of vitamin E prior to analysis. Therefore, the purposes of this project were 1) to determine the stability of vitamin E in refrigerated and frozen porcine liver and serum and 2) to evaluate the effects of red blood cell (RBC) hemolysis on porcine serum vitamin E concentrations. Porcine liver and nonhemolyzed serum were collected and stored refrigerated or frozen. Samples were analyzed for vitamin E immediately or on days 2, 3, 7, or 14. In addition, porcine RBCs were added to normal serum at concentrations from 1 × 106 to 1 × 109 RBC/ml and hemolyzed by freeze-thaw prior to analysis for vitamin E or products of lipid peroxidation.

Myocardial fibrosis associated with previous ingestion of yew (Taxus sp.) in a Holstein heifer evidence for chronic yew toxicity in cattle

Twenty-six 5-month-old Holstein calves were accidentally exposed to discarded branches of yew bushes (Taxus sp.). Several calves were found dead approximately 24 hr after exposure; however, a few calves died several days after exposure. One calf died 18 days after the initial exposure to Taxus sp. and was examined on the farm via necropsy. Gross lesions included ascites, and dilated and flaccid myocardial ventricles. Sections of formalin-fixed heart were submitted to the Indiana Animal Disease Diagnostic Laboratory for histopathologic examination; fresh rumen contents were submitted for toxicologic testing. Histologically, large areas of myocardium were replaced by fibrous connective tissue, suggesting previous myocardial necrosis. Taxus alkaloids were identified in the rumen contents using gas chromatography–mass spectrometry. Based on the clinical history, the gross and histologic lesions, the identification of Taxus alkaloids in the rumen contents, and lack of exposure to other known cardiotoxic agents, yew toxicity was considered the cause of death in this calf. Ingestion of taxines is known to cause acute and subacute toxicity in human beings and animals; however, a chronic clinical course and severe histologic lesions have not been previously associated with yew toxicity. Although only 1 calf was examined, this case suggests that yew toxicity can result in a prolonged clinical course in cattle and can cause histologic myocardial lesions.