In vitro toxicity test of poisonous mushroom extracts with isolated rat hepatocytes.

Effects of poisonous mushroom extracts on isolated rat hepatocytes were studied. Though no significant decrease in the cell viability was observed during the incubation of hepatocytes with the extracts at a concentration of 5% (v/v) of Amanita abrupta, A. gymnopus, and A. virosa caused marked decreases in the intracellular glutathione content in sharp contrast to the extracts of A. volvata and A. flavipes. Comparative toxicity tests were carried out for the effects of the extract of A. abrupta, dl-propargylglycine, and alpha-amanitin. The extract of A. abrupta at a concentration of 1% (v/v) caused a marked decrease in the glycogen content, a noticeable elevation in the phosphorylase alpha activity, and a slight acceleration of lipid peroxidation in the hepatocytes. Although dl-propargylglycine decreased the intracellular glutathione content progressively with the incubation time, a significant effect of the chemical on lipid peroxidation and the glycogen content was observed only after prolonged incubation at a concentration of 5 mM. On the other hand, alpha-amanitin exerted a little effect on the hepatocytes at 1 microM. These results have indicated that the intoxication by the extract of A. abrupta on the hepatocytes might not due to independently each component, dl-propargylglycine and alpha-amanitin, but combined effect of these components or unidentified substances.

[The forensic medical diagnosis of death-cup poisonings]. / Sudebno-meditsinskaia diagnostika otravlenii blednoi pogankoi.

Comparative analysis and evaluation of all modern methods used for medicolegal diagnosis of poisoning with Amanita phalloides are presented. Imperfection of methods used for laboratory diagnosis of such poisonings requires extensive experimental control using the designed method of liquid chromatography for amanitine and phalloidine assay in the cadaveric organs and tissues.

Endocrine hormone abnormalities in Amanita poisoning.

Mushroom poisoning from the genus Amanita is being reported with increasing frequency in the United States. Endocrine hormone abnormalities were investigated in four patients who ingested mushrooms of the Amanita genus ("death cap", "destroying angel"). Marked abnormalities were found in the hormones controlling glucose, calcium, and thyroid homeostasis. Insulin and C-peptide concentrations were elevated at admission, indicating that the hypoglycemia associated with Amanita poisoning may not be solely secondary to hepatic failure. Serum calcitonin concentrations were elevated in conjunction with hypocalcemia. Parathyroid hormone concentrations (both the carboxyl- and amino-terminal assays) increased with time, but began returning to baseline as the hypocalcemia disappeared. The thyroxine concentrations were depressed in all four patients, and triiodothyronine (T3) concentrations were undetectable in three patients. Thyroid-stimulating hormone concentrations were never elevated, reflecting an unresponsive pituitary-hypothalamic axis to the development of hypothyroidism or a euthyroid-illness syndrome.

Hepatotoxic action of a poisonous mushroom, Amanita abrupta in mice and its toxic component.

An aqueous extract of a poisonous mushroom, Amanita abrupta was injected intraperitoneally into male ICR mice and the acute effects on the liver were studied. Contents of serum glucose and liver glycogen decreased to 60% and 10% of the control levels, respectively, 6 h after injection. Activities of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase increased to 3- and 8-fold, respectively, 12 h after injection, and the elevated activities were maintained up to 24 h. Activities of the hepatic drug metabolizing enzymes were also reduced 15 h after injection. Histological examination demonstrated massive liver cell necrosis and disappearance of glycogen granules in the liver of the treated animals. Two amino acids, L-2-amino-4-pentynoic acid and L-2-amino-4,5-hexadienoic acid were identified in the mushroom extract. The former caused similar biochemical effects to those of the mushroom extract.

[Sterols from Basidiomycetes]. / Steroli da Basidiomiceti.

Sterols of three Basidiomycetes were determined. The main sterol was ergosta-5,7,22-trien-3 beta-ol, accompanied by other closely related sterols. Cholesterol was found only in trace amounts.

[Amanita virosa peptides: viroidin and viroisin are more effective than phalloidin for the in vitro protection of actin against the effects of osmic acid]. / Des peptides de Amanita virosa: viroidine et viroisine sont plus efficaces que la phalloidine pour protéger l'actine in vitro contre les effets de l'acide osmique.

Virotoxins are a group of monocyclic peptides recently identified in the deadly mushroom Amanita virosa by Faulstich and coll. We found that two of these peptides, which have a methyl sulfonyl group, namely viroidin and viroisin are very effective to protect F-actin against oxidative degradation by osmium tetroxide in vitro. Their desoxo analogs, which have a methyl sulfoxyde group instead of methyl sulfonyl are less active, therefore there exists a relationship between the chemistry of the sulfur group and the activity of the peptides.

Selection for resistance to phallolysin, a cytolytic toxin from the death-cap mushroom (Amanita phalloides).

Phallolysin-resistant variants are readily selected from unmutagenized populations of mouse fibroblasts. They exhibit partial cross-resistance to wheat germ agglutinin, a lectin with a saccharide specificity similar to phallolysin. These results are consistent with phallolysin acting by binding to cell surface glycoprotein receptors rather than by interacting directly with lipid components of the membrane.

[In vivo and in vitro effects of peptide extracts from Amanita virosa]. / Effets in vivo et in vitro de peptides extraits de Amanita virosa.

Cyclic peptides from the deadly mushroom Amanita virosa has been separated by methanolic extraction and chromatography on Sephadex. Three groups of peptides have been obtained: virotoxins, amaninamide and phalloidin. Virotoxins has been separated in two fractions named virotoxins A and virotoxins B. We have studied the properties of these two fractions of F actin in vitro and on mice in vivo. Our results show that virotoxins A and B protect F actin in vitro against chaotropic ions, depolymerization by DNAse I or cytochalasin B and heat denaturation. Virotoxins A and B increase the rate of polymerization of F actin. Virotoxins A and B are toxic compounds which produce hemorrhagic necrosis of liver that have been observed in detail by electron microscopy. In general, our in vitro and in vivo results show that virotoxins exhibit the same effect as phalloidin on F actin.

A rapid radioimmunoassay, using a nylon support, for amatoxins from Amanita mushrooms.

A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.

Mushrooms and madness. Hallucinogenic mushrooms and some psychopharmacological implications.

This article reviews the major hallucinogenic fungi both for their historical as well as neurochemical import. Despite voluminous literature on them, relatively little study has focused on psilocybin related substances that could relate to forms of psychotic illness. Some metabolic pathways are reviewed which illustrate the need for more study of indole compounds such as baeocystin.

Amaninamide, a new toxin of Amanita virosa mushrooms.

Amaninamide, a toxin closely related to the family of amatoxins, was found exclusively in Amanita virosa mushrooms. It differs from the well known toxin alpha-amanitin in that it lacks the 6'-hydroxyl group of the tryptophan unit, and from the toxin amanin found in Amanita phalloides by the presence of a carboxamide group instead of a carboxylic acid groups.

Determination of alpha-, beta-, and gamma-amanitin by high performance thin-layer chromatography in Amanita phalloides (Vaill. ex Fr.) secr. from various origin.

A fast, sensitive high performance thin-layer chromatographic method for the determination of alpha-, beta-, and gamma-amanitin in crude, methanolic extracts of Amanita phalloides is described. The limit of detection is 50 ng of each amanitin. With this method amanitin was determined in 24 pooled samples of Amanita phalloides, collected between 1970 and 1977 in Germany and Switzerland. The total amanitin content varied between 2010 and 7300 mg/kg dry weight and the average value was 4430 mg/kg of which 43% was alpha-amanitin, 49% beta-amanitin and 8% gamma-amanitin. The origin of the fungi hardly influenced their amanitin content: in samples collected during the same year at different sites it fluctuated within a factor of 1.7. The amanitin content of samples from the same site, but collected in different years, maximally varied within a factor of 3.7. The partial decomposition of amanitins during prolonged storage of the lyophilized samples undoubtedly contributed to this variation. Phalloidin, which was determined by conventional thin-layer-chromatography, could not be detected in a sample from 1970, whereas its concentration in material collected during 1977 amounted to 2400 mg/kg dry weight. The toxicity of the samples (LD50 of lyophilized defatted methanolic extracts intravenously for mice) varied within a factor of 2.5.