Amanitins: The Most Poisonous Molecules of the Fungal World.

Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and certain groups of animals) stand out. Amatoxin-containing mushrooms and the poisonings caused by them stand out from the higher fungi, the mushrooms. There are already historical data and records about such poisonings, but scientific research on the responsible molecules began in the middle of the last century. The goals of this review work are as follows: presentation of the cosmopolitan mushroom species that produce amanitins (which are known from certain genera of four mushroom families), an overview of the chemical structure and specific properties of amanitins, a summary of the analytical methods applicable to them, a presentation of the "medical history" of poisonings, and a summary of the therapeutic methods used so far. The main responsible molecules (the amanitins) are bicyclic octapeptides, whose structure is characterized by an outer loop and an inner loop (bridge). It follows from the unusual properties of amanitins, especially their extreme stability (against heat, the acidic pH of the medium, and their resistance to human, and animal, digestive enzymes), that they are absorbed almost without hindrance and quickly transported to our vital organs. Adding to the problems is that accidental consumption causes no noticeable symptoms for a few hours (or even 24-36 h) after consumption, but the toxins already damage the metabolism of the target organs and the synthesis of nucleic acid and proteins. The biochemical catastrophe of the cells causes irreversible structural changes, which lead to necrotic damage (in the liver and kidneys) and death. The scientific topicality of the review is due to the recent publication of new data on the probable antidote molecule (ICR: indocyanine green) against amanitins. Further research can provide a new foundation for the therapeutic treatment of poisonings, and the toxicological situation, which currently still poses a deadly threat, could even be tamed into a controllable problem. We also draw attention to the review conclusions, as well as the mycological and social tasks related to amanitin poisonings (prevention of poisonings).

Reverse-phase/phenylboronic-acid-type magnetic microspheres to eliminate the matrix effects in amatoxin and phallotoxin determination via ultrahigh-performance liquid chromatography-tandem mass spectrometry.

In this study, we present the preparation of a new reverse-phase/phenylboronic-acid (RP/PBA)-type mixed-mode magnetic solid-phase extraction (MSPE) adsorbent for use in the cleanup of amatoxin- and phallotoxin-containing samples intended for ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Further, the RP/PBA magnetic microspheres have phenyl and phenylboronic acid groups on their surfaces that selectively adsorb amatoxins and phallotoxins through hydrophobic, π-π, and boronate affinity, significantly reducing matrix effects in UPLC-MS/MS analysis. After systematic optimization, all the standard calibration curves expressed satisfactory linearity (r > 0.9930), limits of detection (0.3 µg/kg), and recovery (97.6%-114.2%). Compared with other reported methods, this method also has the advantages of simple, fast, and efficient operation using relatively small amounts of the MSPE adsorbent. Furthermore, the method was successfully applied in a poisoning incident caused by Lepiota brunneoincarnata Chodat & C. Martín ingestion.

Effectiveness of Fractionated Plasma Separation and Absorption as a Treatment for Amanita Phalloides Poisoning.

BACKGROUND Fractionated plasma separation and absorption (FPSA) is an extracorporeal liver support method that detoxifies accumulated toxins. There are limited data of its use in the treatment of Amanita phalloides intoxication. The objective of this study was to investigate whether FPSA before liver transplantation improves patients' short-term post liver transplantation survival in Amanita phalloides poisoning. MATERIAL AND METHODS The study population consisted of ten patients who had liver transplantation (LT) due to acute liver failure (ALF) caused by Amanita phalloides poisoning. Six patients were treated with FPSA before liver transplantation. All the patients who were started on FPSA were also placed on the liver transplantation list according to emergent liver transplantation criteria. RESULTS Patients treated with FPSA were in a more severe clinical condition presenting in higher mean MELD, total bilirubin, INR and ammonia along with more frequent hypoglycemia and hepatic encephalopathy grade 3/4. FPSA group had longer mean waiting time on the recipient list (3.5 vs. 1.25 days) but inferior thirty-day survival rate (16.5% vs. 100%). CONCLUSIONS When conservative medical modalities are ineffective, the only treatment for Amanita phalloides poisoning is a liver transplant. Although FPSA treated patients had inferior post-LT survival, FPSA was found to prolong the pre surgical waiting time for critically ill patients, consequently giving a chance of life-saving procedure.

Fabrication of a biomimetic adsorbent imprinted with a common specificity determinant for the removal of α- and ß-amanitin from plasma.

α-Amanitin and ß-amanitin are the main toxins of mushroom poisoning. The application of traditional non-selective adsorbents is not satisfactory in clinical treatment of amanita mushroom poisoning due to lack of specificity adsorption capability of these adsorbents toward amanitin toxins. In the current work, we introduce a novel molecularly imprinted biomimetic adsorbent based on a ligand specificity determinant through surface imprinted strategy. Owing to the expensive price of the amanitin sources, we selected a typical common moiety of α, ß-amanitin as specificity determinant to synthesize a template necessary for the preparation of molecularly imprinted polymers (MIPs). Computer simulation was used to initially select acidic methacrylic acid (MAA) and basic 4-vinyl pyridine (4-VP) together as functional monomers. The experiments further demonstrated that the synergistic interaction of MAA and 4-VP played a primary role in the recognition of α, ß-amanitin by MIPs. By means of batch and packed-bed column experiment and the hemocompatibility evaluation, the resultant biomimetic adsorbent has been proved to be capable of selectively removing α, ß-amanitin and possess good hemocompatibility. This novel adsorbent has great potential to find application in human plasma purification.

Molecular identification of poisonous mushrooms using nuclear ITS region and peptide toxins: a retrospective study on fatal cases in Thailand.

Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

Cytotoxic constituents of Amanita subjunquillea.

As part of our systematic study of Korean toxic mushrooms, we have investigated the constituents of Amanita subjunquillea. The column chromatographic separation of the MeOH extract of A. subjunquillea led to the isolation of four ergosterols, two cerebrosides and four cyclopeptides. Their structures were determined by spectroscopic methods to be (22E,24R)-5alpha,8alpha-epidioxyergosta-6,9,22-triene-3beta-ol (1), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (2), (22E,24R)-5alpha,6alpha-epoxyergosta-8,22-diene-3beta,7beta-diol (3), (24S)-ergost-7-en-3beta-ol (4), 8,9-dihydrosoyacerebroside I (5), soyacerebroside I (6), beta-amanitin (7), phalloin (8), alpha-amanitin (9), and phalloidin (10). The compounds 1-6 and 8 were isolated for the first time from this mushroom. The isolated compounds were evaluated for the cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT15 cells. Compound 9 exhibited significant cytotoxic activity against A549, SK-OV-3, SK-MEL-2 and HCT15 with ED(50) values of 1.47, 0.26, 1.57 and 1.32 microM, respectively.

[Culture conditions and analysis of amanitins on Amanita spissa].

Isolate of Amanita spissa was obtained from basidiome stipe material collected from environment. It could utilize a broad range of carbon and nitrogen resources. Study on the influence of different conditions for solid culture was carried out. Optimal culture conditions were at 28 degrees C, pH6, in the dark. A. spissa was then fermentated in liquid culture for more mycelia. In flask and Airlift/ff bioreactor, maximum dry mycelia weight of A. spissa reached 0.893 g/L and 2.33 g/L, respectively. Mycelia obtained from solid culture and Airlift/ff bioreactor were then analyzed by HPLC. The results showed that mycelia from both cultures contained amatoxins but no phallotoxins. alpha-Amanitin in mycelia reached 26.02 microg/DWg under solid culture condition, and 15.25 microg/DWg under liquid culture condition. The amanitins were also confirmed by bud-inhibited assay. The results revealed that the effect of amanitin on mung bean cell was identical to that of authentic amanitins. This work suggests that it is possible to produce amatoxin by liquid culturing of A. spissa.

Production and characterization of Amanitin toxins from a pure culture of Amanita exitialis.

Amanita exitialis Zhu L. Yang and T.H. Li is a lethal mushroom species recently isolated in Guangdong Province, China. In this report, a pure culture of this species was obtained for the first time. To confirm the identity of the pure culture, the internal transcribed spacer regions of the nuclear ribosomal DNA of the pure culture and of a typical fruiting body of the species were sequenced and compared. Further, amatoxins produced by pure cultures were analyzed and characterized by high-performance liquid chromatography and mass spectrometry analysis. The results showed that the pure cultures produced 728.3 +/- 43.8 microg g(-1) (dry matter) of alpha-Amanitin and 60.0 +/- 20.7 microg g(-1) (dry matter) of beta-Amanitin, respectively, a yield which is about 10% of that produced by fruiting bodies.

Feasibility of immunodiagnostic devices for the detection of ricin, amanitin, and T-2 toxin in food.

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Detection and identification of amanitins in the wood-rotting fungi Galerina fasciculata and Galerina helvoliceps.

More than 600 strains of wood-rotting fungi were screened for the detection of amanitins. Three strains of Galerina fasciculata and 18 strains of Galerina helvoliceps contained amanitins. These strains contained mainly alpha- and beta-amanitins in the native fruit bodies, while alpha- and gamma-amanitins were found in liquid-cultured mycelia. Purified amanitins were confirmed by their chromatographic profiles, spectra (UV, Fourier transform infrared, and atmospheric ionization mass), cytotoxicity for mammalian cell lines (3T3 and SiHa), and inhibitory effects on RNA polymerase II. The results revealed that the purified amanitin fractions from these species are identical to authentic amanitins and suggest that these two species must be handled as poisonous mushrooms.

Polyclonal amanitin-specific antibodies: production and cytoprotective properties in vitro.

The amanitins found in several mushroom species are responsible for many deaths every year. Based on its successful application to cardiac glycoside overdose, immunotherapy could be applicable to amanitin toxicity. Therefore, we produced polyclonal amanitin antibodies by immunizing rabbits with a novel conjugate of alpha-amanitin. Purified antibodies had an average association constant for alpha-amanitin of 1.3 x 10(9) M-1. A partially protective effect of the antibodies against amanitin toxicity in vitro in Chang cells was evident at a molar ratio of antibody binding sites to alpha-amanitin of 4:1. Together with reported studies in vivo, these investigations indicate the potential of immunotherapy for amanitin poisoning.

Periodate oxidation products derived from methylated alpha-amanitin: evidence for distinct aldehydic and non-aldehydic forms.

Amatoxins are cyclic octapeptides which can be purified from various mushroom species. They have found widespread use due to their potent inhibition of eukaryotic RNA polymerase II. In the course of our efforts to prepare additional semisynthetic derivatives of the amatoxin, alpha-amanitin, we examined the products formed through the periodate oxidation of 6'-O-methyl-alpha-amanitin. Periodate oxidation under conditions of near-neutral pH yielded two chemically similar, yet chromatographically separable, products. On the basis of their proton NMR spectra and their lack of reactivity with sodium chlorite these products did not contain a free aldehydic group. However, both forms were interconvertible in aqueous neutral solution and could be converted to the same product, 6'-O-methyldemethyl-gamma-amanitin, through reduction with sodium borohydride. Periodate oxidation under mildly acidic conditions generated a single product which has properties of a free aldehyde. It exhibited a proton NMR spectrum with signals characteristic of an aliphatic aldehyde and was readily oxidized to the carboxylic acid with sodium chlorite. Conditions have been defined to synthesize the free aldehyde derivative of 6'-O-methyl-alpha-amanitin in generous yield to provide a precursor for oxidation and further derivatization.

Improved synthesis and purification of methylated amanitins using diazomethane.

We have examined the conditions of methylating alpha-amanitin with diazomethane with the intent of producing 6'-O-methyl-alpha-amanitin (meAMA). Under the appropriate conditions meAMA was afforded as the sole product in nearly quantitative yield. By exceeding the stoichiometries designed for optimal meAMA synthesis, a dimethylated amanitin, 1'-N-, 6'-O-dimethyl-alpha-amanitin (dimeAMA), was also produced. Both products were characterized, following HPLC, by ultraviolet and n.m.r. spectroscopy. Based upon their inhibitory potential against wheat germ RNA polymerase II, apparent dissociation constants of 4.3 nM and 5.4 nM were estimated for meAMA and dimeAMA, respectively.

Physical properties and function of phallolysin.

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The toxic peptides from Amanita mushrooms.

The results of 50 years of effort in the chemistry of Amanita toxins are reviewed. The phallotoxins, fast acting components, but not responsible for fatal intoxications after ingestion, are bicyclic heptapeptides. They combine with F-actin, stabilizing this protein against several destabilizing influences. The virotoxins likewise fast acting are monocyclic heptapeptides. The amatoxins which are the real toxins lead to death within several days by inhibiting the enzymatic synthesis of m-RNA. They are bicyclic octapeptides. The structures of all of these compounds are described, as well as conformations, chemical reactions and modification, syntheses and correlations between structures and biological activities.

alpha-Amanitin inhibits the oxidation of long chain fatty acids in mouse liver.

It was found that the injection of the extract of Amanita virosa mushroom into mice resulted in the inhibition of long chain fatty acid oxidation by liver mitochondria. The inhibition principle was identified as alpha-amanitin. The inhibition was observed several hours after the injection of alpha-amanitin at the level of about 0.2 mg/kg, which corresponded to LD50. Activities of carnitine acyltransferases, acyl-CoA dehydrogenases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase remained constant. Using fatty acids with various carbon chain lengths as substrates, the oxidation of long chain fatty acids was markedly inhibited with accompanying disappearance of the carnitine-dependent component. The decreases in the oxidation of medium and short chain fatty acids were less marked. It is suggested, therefore, that the site of the inhibition is the transport of long chain acylcarnitines. Peroxisomal beta-oxidation was not affected by the alpha-amanitin treatment.

[The early removal of amatoxins in the treatment of amanita phalloides poisoning (author's transl)]. / Die frühzeitige Elimination der Amanta Toxine in der Therapie der Knollenblätterpilzvergiftung.

In a 3 year period (1975-77) 50 patients have been admitted to the I.C.U. of Polyclinic Hospital of Milan for poisoning from mushrooms of Amanita genus. In 47 cases the diagnosis was confirmed "a posteriori" by serum or urinary detection of amtoxins and/or by clinical evidence of typical liver injury. Besides the symptomatologic support, the therapeutic treatment included combined removal procedures, such as peritoneal dialysis, plasmapheresis, forced diuresis. The detection by radioimmunoassay of amatoxins [6] in the serum and in the urine of these patients proves that this therapeutic treatment can be effective within about 36 hours from ingestion time. The intensive medical care and the removal approach yielded as the whole favourable results in our patients (overall mortality was 6 patients, i.e. 12,7%). It should moreover be emphasized that of the 35 patients, who had been treated with early removal techniques, 12 with ascertained amanita poisoning, had neither clinical nor biochemical evidence of hepatic damage; 14 had a moderate liver damage; 9 experienced a severe liver failure and hepatic coma occurred in 4 of the latter. These poor results can be ascribed to the severity of the poisoning as well as to a peculiar kinetic of amatoxins in each subject.

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.