Mushroom Amanita Phalloides Research Articles

Salidroside Inhibits α-Amanitin-Induced AML-12 Cell Apoptosis via the Regulation of PINK1/Parkin-Mediated Mitophagy and Mitochondrial Function.

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.

The effects of CDP-choline treatment in Amanita phalloides mushroom toxicosis

Amanita phalloides poisoning is known to be the most fatal case among mushroom poisoning cases. Its main mechanism of toxicity is that it leads to cell death by the irreversible binding of its toxins to the DNA-dependent RNA polymerase II enzyme. This study was planned to analyze the effects of the CDP-choline molecule on Amanita phalloides mushroom poisoning cases.The extract of the Amanita phalloides mushroom was taken and intraperitoneally administered to male Wistar Albino rats at a dose of 0.3 g/kg. In the experiment phase, the rats were divided into three groups of CDP-choline treatment according to the doses of 100 mg/kg, 250 mg/kg, and 500 mg/kg, and one control group was administered a 1 ml/kg dose of 0.9% isotonic NaCl solution. The treatments were then administered intraperitoneally at the 2nd hour, and at the 6th hour, the rats were sacrificed. The degree of damage in the liver and kidney tissues of the rats was evaluated histopathologically.It was concluded that CDP-choline reduced or prevented the damage that occurred in the liver significantly and dose-dependently in the toxicosis picture caused by Amanita phalloides, and it showed a tendency to lower or prevent the damage in the kidney, albeit not significantly.

Distribution changes of the toxic mushroom Amanita phalloides under climate change scenarios and its potential risk over indigenous communities in Mexico

Amanita phalloides is a native European deathly ectomycorrhizal mushroom that was introduced to North America and has been expanding its distribution during the last decades. This species is morphologically similar to wild edible mushrooms and if its distribution expands to Mexico, it could represent a risk in terms of food security for local communities. The aim of this study was to evaluate the potential climatic suitability that exists for A. phalloides in North America and overlay it with the distribution of mycophilic communities in Mexico under a baseline climatic scenario and climate change scenarios. To find climatic suitability we modeled its potential distribution with the algorithm that had the best predictive power after pilot test (MaxEnt) using species presences and eight climatic variables chosen with biological and statistical criteria. We worked with CanESM5 because it is one of the best models to simulate climate in North America and SSP5-8.5 scenario in order to be consistent with the precautionary principle. Our results suggest that even when the species has not yet been registered in Mexico, when using European records to model, this country presents 33.61% of climatic suitability for this species under the baseline scenario, potentially affecting about 70% of indigenous communities which are the main consumers of edible mushrooms. Under climate change scenarios, an increase in climatic suitability is expected in Mexico, while decreases are expected in United States and Canada. When using North American records to model, almost no climatic suitability is found in Mexico; however, the implementation of warning campaigns in Mexico is still needed.

Pangenomics of the death cap mushroom Amanita phalloides, and of Agaricales, reveals dynamic evolution of toxin genes in an invasive range

The poisonous European mushroom Amanita phalloides (the “death cap”) is invading California. Whether the death caps’ toxic secondary metabolites are evolving as it invades is unknown. We developed a bioinformatic pipeline to identify the MSDIN genes underpinning toxicity and probed 88 death cap genomes from an invasive Californian population and from the European range, discovering a previously unsuspected diversity of MSDINs made up of both core and accessory elements. Each death cap individual possesses a unique suite of MSDINs, and toxin genes are significantly differentiated between Californian and European samples. MSDIN genes are maintained by strong natural selection, and chemical profiling confirms MSDIN genes are expressed and result in distinct phenotypes; our chemical profiling also identified a new MSDIN peptide. Toxin genes are physically clustered within genomes. We contextualize our discoveries by probing for MSDINs in genomes from across the order Agaricales, revealing MSDIN diversity originated in independent gene family expansions among genera. We also report the discovery of an MSDIN in an Amanita outside the “lethal Amanitas” clade. Finally, the identification of an MSDIN gene and its associated processing gene (POPB) in Clavaria fumosa suggest the origin of MSDINs is older than previously suspected. The dynamic evolution of MSDINs underscores their potential to mediate ecological interactions, implicating MSDINs in the ongoing invasion. Our data change the understanding of the evolutionary history of poisonous mushrooms, emphasizing striking parallels to convergently evolved animal toxins. Our pipeline provides a roadmap for exploring secondary metabolites in other basidiomycetes and will enable drug prospecting.

Role of Silymarin in Cancer Treatment: Facts, Hypotheses, and Questions.

The flavonoid silymarin extracted from the seeds of Sylibum marianum is a mixture of 6 flavolignan isomers. The 3 more important isomers are silybin (or silibinin), silydianin, and silychristin. Silybin is functionally the most active of these compounds. This group of flavonoids has been extensively studied and they have been used as hepato-protective substances for the mushroom Amanita phalloides intoxication and mainly chronic liver diseases such as alcoholic cirrhosis and nonalcoholic fatty liver. Hepatitis C progression is not, or slightly, modified by silymarin. Recently, it has also been proposed for SARS COVID-19 infection therapy. The biochemical and molecular mechanisms of action of these substances in cancer are subjects of ongoing research. Paradoxically, many of its identified actions such as antioxidant, promoter of ribosomal synthesis, and mitochondrial membrane stabilization, may seem protumoral at first sight, however, silymarin compounds have clear anticancer effects. Some of them are: decreasing migration through multiple targeting, decreasing hypoxia inducible factor-1α expression, inducing apoptosis in some malignant cells, and inhibiting promitotic signaling among others. Interestingly, the antitumoral activity of silymarin compounds is limited to malignant cells while the nonmalignant cells seem not to be affected. Furthermore, there is a long history of silymarin use in human diseases without toxicity after prolonged administration. The ample distribution and easy accessibility to milk thistle—the source of silymarin compounds, its over the counter availability, the fact that it is a weed, some controversial issues regarding bioavailability, and being a nutraceutical rather than a drug, has somehow led medical professionals to view its anticancer effects with skepticism. This is a fundamental reason why it never achieved bedside status in cancer treatment. However, in spite of all the antitumoral effects, silymarin actually has dual effects and in some cases such as pancreatic cancer it can promote stemness. This review deals with recent investigations to elucidate the molecular actions of this flavonoid in cancer, and to consider the possibility of repurposing it. Particular attention is dedicated to silymarin's dual role in cancer and to some controversies of its real effectiveness.

Amperometric Biosensors for L-Arginine Determination Based on L-Arginine Oxidase and Peroxidase-Like Nanozymes

There are limited data on amperometric biosensors (ABSs) for L-arginine (Arg) determination based on oxidases that produce hydrogen peroxide (H2O2) as a byproduct of enzymatic reaction, and artificial peroxidases (POs) for decomposition of H2O2. The most frequently proposed Arg-sensitive oxidase-based ABSs contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Therefore, the construction of a one-enzyme ABS for Arg analysis is a practical problem. In the current work, fabrication, and characterization of three ABS types for the direct measurement of Arg were proposed. L-arginine oxidase (ArgO) isolated from the mushroom Amanita phalloides was co-immobilized with PO-like nanozymes (NZs) on the surface of graphite electrodes. As PO mimetics, chemically synthesized NZs of CeCu (nCeCU) and NiPtPd (nNiPtPd), as well as green-synthesized hexacyanoferrate of copper (gCuHCF), were used. The novel ABSs exhibited high sensitivity and selectivity to Arg, broad linear ranges and good storage stabilities. Two ABSs were tested on real samples of products containing Arg, including the pharmaceutical preparation “Tivortine”, juices, and wine. A high correlation (R = 0.995) was demonstrated between the results of testing “Tivortine” and juice using nCeCU/GE and nNiPtPd/GE. It is worth mentioning that only a slight difference (less than 1%) was observed for “Tivortin” between the experimentally determined content of Arg and its value declared by the producer. The proposed ArgO-NZ-based ABSs may be promising for Arg analysis in different branches of science, medicine, and industry.

The Role of Therapeutic Plasma Exchange in the Treatment of Childhood Intoxication: A Single-Center Experience.

Therapeutic plasma exchange is used to treat neurologic, hematological, renal, and autoimmune diseases with a known or suspected etiopathogenesis. However, there is incomplete understanding of the use of therapeutic plasma exchange in pediatric cases of intoxication. This study investigated 5 years of experience with therapeutic plasma exchange to treat intoxication cases. A retrospective, case series, single-center study. PICU of Baskent University, Dr. Turgut Noyan Teaching, and Medical Research Center Hospital in Adana, Turkey. Fourteen patients diagnosed with intoxication who underwent therapeutic plasma exchange between January 2013 and January 2018. None. Data pertaining to 14 patients, including their medical history (exposure to drugs/toxicants), demographics, initial presentation, and severity of clinical symptoms (requirement of mechanical ventilation, Glasgow Coma Scale score, and the pediatric severity of illness score [Pediatric Logistic Organ Dysfunction] were retrospectively reviewed. The most common indication for therapeutic plasma exchange was multiple drug intoxication, followed by amitriptyline, Amanita phalloides mushroom, carbamazepine, mercury, verapamil, and botulism. All patients underwent therapeutic plasma exchange and two patients underwent hemodialysis before therapeutic plasma exchange. There was no mortality or complications related to the therapeutic plasma exchange procedure. Clinical improvement was observed after therapeutic plasma exchange in 13 of the 14 patients; one patient with verapamil intoxication died. Therapeutic plasma exchange appears to be safe and effective for treating pediatric cases of intoxication, including multidrug and amitriptyline intoxication, and is associated with significant recovery in the majority of severely affected patients. Treatment of intoxication with therapeutic plasma exchange should be guided primarily by the properties of the causative toxic substances/drugs, and consideration of patient age, the severity of clinical symptoms, Pediatric Logistic Organ Dysfunction score and response to initial supportive and antidotal treatment.

Prognosis and treatment options in cases of acute liver failure caused by mushroom poisoning due to Amanita phalloides

Poisoning due to mushroom ingestion is a relatively rare but deadly cause of acute liver failure (ALF). Consumption of the poisonous mushroom Amanita phalloides, also known as ‘death cap’, is one of the most common causes of mushroom poisoning worldwide, being involved in the majority of human fatalities caused due to mushroom ingestion. A major portion of the liver damage due to Amanita phalloides is related to powerful toxins known as amanitins, which cause impairment in protein synthesis and subsequent cell necrosis by the inhibition of RNA polymerase II. Initially the presentation is that of an asymptomatic lag phase, followed by gastrointestinal symptoms and hepato-renal involvement. Amatoxin poisoning may progress into fulminant hepatic failure and eventually death if liver transplantation is not performed. It is based on a careful assessment of history of type and duration of mushroom ingestion, as well as the clinical manifestations. Diagnosis can be confirmed by laboratory tests measuring urinary amatoxin levels and identification of the mushroom. Although N-Acetyl Cysteine and Penicillin-G have proven to be effective therapeutic agents, Orthotopic Liver Transplantation (OLT) or Auxiliary Partial Orthotopic Liver Transplantation (APOLT) is the only treatment option for most of the cases carrying a poor prognosis.

Total Synthesis of the Death Cap Toxin Phalloidin: Atropoisomer Selectivity Explained by Molecular-Dynamics Simulations.

Phallotoxins and amatoxins are a group of prominent peptide toxins produced by the death cap mushroom Amanita phalloides. Phalloidin is a bicyclic cyclopeptide with an unusual tryptathionin thioether bridge. It is a potent stabilizer of filamentous actin and in a fluorescently labeled form widely used as a probe for actin binding. Herein, we report the enantioselective synthesis of the key amino acid (2S,4R)-4,5-dihydroxy-leucine as a basis for the first total synthesis of phalloidin, which was accomplished by two different synthesis strategies. Molecular-dynamics simulations provided insights into the conformational flexibility of peptide intermediates of different reaction strategies and showed that this flexibility is critical for the formation of atropoisomers. By simulating the intermediates, rather than the final product, molecular-dynamics simulations will become a decisive tool in orchestrating the sequence of ring formation reactions of complex cyclic peptides.

The histopathological features of the severe intoxication with amatoxin (toxin contained in Amanita phalloides mushrooms)

Eating mushrooms known to contain amatoxin is fraught with serious complications. The analysis of the relevant literature publications revealed no article with the description of the histological picture of the internal organs in the subjects intoxicated with amatoxin. It is known, however, that such poisoning is associated with the severe irreversible injuries to all intracellular protein structures the character of which depends on time. Specifically, acute amatoxin intoxication produces the well apparent clinical picture within 6 days after intake of the poison. It is characterized by acute renal and hepatic insufficiency in the combination with the injury to the conducting system of heart and the myocrardium itself. Thereafter, the disseminated intravascular coagulation (DIC) syndrome developed accompanied by the signs of progressive tissue hypoxia that ended in death on day 9. The histological study has demonstrated necrotic foci in the liver and oedematous hepatic stroma. Kidneys underwent multiple hemorrhages, necrosis of convoluted tubules and well apparent hydropic protein dystrophy of their epithelium. The adrenal glands showed up signs of necrosis and hemorrhage. It is concluded that poisoning with mushrooms (amatoxin) should be regarded as the most probable cause of the condition requiring differential diagnostics between acute gastroenteritis and renal insufficiency.

Affinity capillary electrophoresis and quantum mechanical calculations applied to investigation of [Gly6 ]-antamanide binding with sodium and potassium ions.

ACE in a free solution and quantum mechanical density functional theory have been applied to the investigation of interactions of glycine-6-antamanide ([Gly6 ]AA), a synthetic derivative of cyclic decapeptide antamanide isolated from the highly poisonous mushroom Amanita phalloides, with sodium or potassium ions in methanol. First, from the dependence of effective electrophoretic mobility of [Gly6 ]AA on Na+ or K+ ions concentration in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pHMeOH 7.8, containing 0-50 mM NaCl or 0-40 mM KCl), the apparent binding (stability) constants of [Gly6 ]AA-Na+ and [Gly6 ]AA-K+ complexes were evaluated as 26 ± 1 and 14 ± 1L/mol, respectively. The employed ACE method included correction of the effective mobilities measured at ambient temperature and at variable ionic strength of the BGEs to the mobilities related to the reference temperature 25°C and to the constant ionic strength 10 mM. Second, the interaction energies of the [Gly6 ]AA-Na+ and [Gly6 ]AA-K+ complexes (-466.3 and -345.2 kJ/mol, respectively) and the structural details of these complexes, such as position of the Na+ and K+ ions in the cavity of the [Gly6 ]AA molecule and the interatomic distances within these complexes, were determined by the density functional theory calculations.

Evaluation and comparison of alpha- and beta-amanitin toxicity on MCF-7 cell line

Alpha- and beta-amanitins are the main toxins of the poisonous Amanita phalloides mushroom. Although there are many studies available concerning alpha-amanitin, there are limited data about beta-amanitin in the literature. Therefore, this study is aimed at comparing the toxic effects of alpha- and beta-amanitin on the MCF-7 cell line. The alpha- and beta-amanitins used for this research were purified from Amanita phalloides by preparative high-performance liquid chromatography. The MCF-7 breast cancer cell line was used, and specific concentrations of the toxins (100, 10, 1, 0.1, and 0.01 μg/mL) were applied to the cells. The MTT test was performed to determine the level of toxicity, and the quantity of protein in the cell was measured using the biuret test. The aLpha-amanitin showed a higher toxicity at 36 h, while the highest inhibition of protein synthesis by the beta-amanitin was observed at 24 h. It was shown that the beta-amanitin may be responsible for toxicity, like alpha-amanitin, in Amanita phalloides mushroom poisoning. The early inhibition of protein synthesis for beta-amanitin might be useful for future experiments and research.

A rapid test for toxins from Amanita phalloides mushrooms

A rapid test for amanitins is proposed on the basis of their complexation with ethydium bromide leading to an intensively fluorescent adduct; the fluorescence band of the product has a shorter wavelength than that of ethydium bromide, which ensures the high specificity of the analysis. The fluorescence intensity in the system of ethydium bromide — amanitin grows linearly with the concentration of amanitin in the range 0.2–1.7 μM at the constant concentration of ethydium bromide, 0.6 μM. The detection limit is 1 μM, the time of the development of the analytical signal is up to 5 s.

Influence of silibinin on the permeability of primary cultured cell membranes

Silibinin, an antihepatotoxic flavanolignane present in Silybum marianum (L.) Gaertn in concentrations greater than 20 μM, inhibits the permeability of parenchymal liver cells to amino acid and nucleotide precursors in vitro by interacting unspecifically with the protein components of the plasma membrane. These findings demonstrate that the hepatoprotective activity of silibinin could be explained, at least in part, by its ability to stabilize the membrane of hepatocytes. This stabilizing effect may find a beneficial application in the therapy of various hepatic disorders especially those involving abnormal fragility of the hepatocyte membrane. This work pinpoints the plasma membrane of parenchymal liver cells as the target for silibinin action and confirms the mechanism of action against the toxic peptide, α-amanitin, from the death cup mushroom Amanita phalloides.

A new highly toxic protein isolated from the death capAmanita phalloidesis an l‐amino acid oxidase

A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21-32]. The physico-chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin-induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l-amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l-amino acid oxidase.

Early Molecular Adsorbents Recirculating System Treatment of Amanita Mushroom Poisoning

Acute poisoning due to ingestion of hepatotoxic Amanita sp. mushrooms can result in a spectrum of symptoms, from mild gastrointestinal discomfort to life-threatening acute liver failure. With conventional treatment, Amanita phalloides mushroom poisoning carries a substantial risk of mortality and many patients require liver transplantation. The molecular adsorbent recirculating system (MARS) is an artificial liver support system that can partly compensate for the detoxifying function of the liver by removing albumin-bound and water-soluble toxins from blood. This treatment has been used in acute liver failure to enable native liver recovery and as a bridging treatment to liver transplantation. The aim of the study is to evaluate the outcome of 10 patients with Amanita mushroom poisoning who were treated with MARS. The study was a retrospectively analyzed case series. Ten adult patients with accidental Amanita poisoning of varying severity were treated in a liver disease specialized intensive care unit from 2001 to 2007. All patients received MARS treatment and standard medical therapy for mushroom poisoning. The demographic, laboratory, and clinical data from each patient were recorded upon admission. The one-year survival and need for liver transplantation were documented. The median times from mushroom ingestion to first-aid at a local hospital and to MARS treatment were 18 h (range 14-36 h) and 48 h (range 26-78 h), respectively. All 10 patients survived longer than one year. One patient underwent a successful liver transplantation. No serious adverse side-effects were observed with the MARS treatment. In conclusion, MARS treatment seems to offer a safe and effective treatment option in Amanita mushroom poisoning.

Progress of acute liver failure and indications for liver transplantation

To analyse the course of acute liver failure and the indications for liver transplantation. In 16 patients who developed acute liver failure between July 1992 and July 1997 the indications for liver transplantation and total survival time were analysed retrospectively. Intensive medical care (ventilation, dialysis) was necessary in 60% of the patients to bridge organ failure until liver transplantation. In 14 patients liver transplantation was indicated. Eleven of the patients, with a minimal Quick value of less than 10%, presented with the prognostically poorest London criteria. In two patients the combination of three additional criteria of the London classification led to liver transplantation being indicated. In one patient with Wilson's disease the transplantation was undertaken despite good clotting factor synthesis, because an encephalopathy occurred. In two patients liver transplantation was not undertaken even though indicated, in one because liver functions improved, in the other because necrotizing pancreatitis had occurred. In another patient, who had been poisoned by eating the mushroom Amanita phalloides, the attempted transplantation was cut short, because intestinal necrosis had developed, and the patient died during the operation. Nine of the eleven patients who had a transplantation and three of the four treated conservatively survived. The 60-day survival rate in this series, achieved in a recently established transplantation programme, is high (12 of 16; 75%), being comparable to those in larger transplantation centres.

A new toxic protein from death cap Amanita phalloides: isolation and study of cytotoxic activity

Cytotoxic protein from fruit body of the death cap Amanita phalloides mushroom, designated as toxophallin, has been isolated and its principal physico-chemical and biological properties have been characterized. This protein possesses molecular mass 55 kDa and isoelectric point at pH 5.7 and is expressed predominantly in the stem of mushroom fruit body. As assessed by cross-linking experiments, toxophallin forms a trimeric complex in buffered solution. It induces apoptosis in several mammalian cells lines (murine L1210 and CCL-64, and human A549) with IC 50 at 0.25–0.45 ug/ml. It also induces DNA fragmentation and morphological changes in the nuclei of target cells (chromatin condensation and fragmentation of nuclei) that are characteristic for apoptosis. Caspase III Inhibitor (Boc-D-fmk) does not inhibit toxophallin-induced apoptotic DNA fragmentation, suggesting that this toxin involves caspase-independent pathway of apoptosis. Besides, it was shown that toxophallin interaction with target cells is not mediated by specific cell surface receptor. Thus, toxophallin is a new toxic protein whose properties distinguish it from other toxic compounds (cyclopeptides and phallolysin) earlier found in the death cap. Keywords: toxic protein, death cap Amanita phalloides , apoptosis.

Free radical reactions might contribute to severe alpha amanitin hepatotoxicity – A hypothesis

Alpha amanitin is a powerful natural hepatotoxin that belongs to the amatoxins isolated from deadly poisonous Amanita phalloides mushroom. The basic molecular mechanism of their toxicity was attributed to inhibition of RNA polymerase II of the eukaryotic cells. At present, the most effective clinical antidote to acute Amanita phalloides mushroom poisoning is silybin, an antioxidant possessing free radical scavenger activity and inhibiting lipid peroxidation, stabilizing membrane structure and protecting enzymes under conditions of oxidative stress. Bearing in mind the biological mechanism of silybin action and the fact that for different amatoxins (alpha, beta, and est. amanitins) does not established straight correlation between their in vivo LD50 and inhibitory constants (Ki) toward RNA polymerase III in vitro determined we supposed some additional toxic effects of these toxins might contribute to their severe hepatotoxicity. Our formerly in vitro experiments demonstrated that alpha amanitin could act either as an antioxidant or as a prooxidant depending on the treatment conditions and toxin concentration. By UV-visible spectroscopy we also shown that alpha amanitin was sensitive to oxidation by a system of lactoperoxidase/H(2)O(2) and assumed formation of free radical toxin intermediates. Having in mind some exogenic compounds including natural toxins can induce increased production of reactive oxygen species (ROS) we suggested similar generation of ROS provoked by alpha amanitin. Our recently in vitro studies have demonstrated that the alpha amanitin could increase superoxide dismutase (SOD) activity and inhibit catalase (CAT) activity to a considerable degree after together incubation of the toxin with any of enzymes. We have also shown that in vitro increased SOD activity was due to superoxide anion radical scavenging activity (SSA) of the toxin. This therefore informed the decision to study the in vivo effect of alpha amanitin on SOD and CAT activity and the level of lipid peroxidation (LPO) products in liver homogenates isolated from mice treated with the toxin. Statistical significant increased level of LPO products was found at the 6th day comparing to the 20th hour after mice treatment with a subletal dose of the toxin. Based on our previous in vitro and present in vivo studies we have made a hypothesize that in vivo during liver accumulation of the toxin it might be transformed to free radical intermediates causing increase in ROS levels. As a result a peroxidative process in hepatocytes might contribute to the severe alpha amanitin hepatotoxicity.

Liver transplantation represents the optimal treatment for fulminant hepatic failure from amanita phalloides poisoning

Amanita phalloides (AP) mushrooms poisoning is a rare but serious occurrence. The main causes of this intoxication are the amatoxines, which inhibit DNA-dependent RNA polymerase II or B. The typical liver and kidney alterations of AP poisoning, consisting of massive hepatic central lobular cell necrosis and acute tubular necrosis of the kidney, are well known [1]. Recognizing the phalloides syndrome allows the institution of effective treatment to avoid possibly fatal complications. The overall prognosis for patients with fulminant hepatic failure (FHF) is poor without transplantation, with survival rates usually reported between 10% and 30% [2]. Medical treatment can prevent complications such as infections and cerebral oedema, which are the most common causes of death. Therapeutic options employed to treat mushroom intoxication include: haemodiaperfusion on activate charcoal, high dosages of penicillin G, oral charcoal, Molecular Adsorbent Recycling System (MARS) and N-acetyl cysteine infusion. When all of them fail, liver transplantation (LT) is the only option [3].When liver dysfunction occurs, transferring the patients to a liver transplant center is crucial to identify appropriate candidates for emergency LT. Indications for LT primarily include medically untreatable and markedly prolonged prothrombin time (PTT), and a constellation of findings such a metabolic acidosis, hypoglycaemia, hypofibrinogenemia and increased serum ammonia, following high serum aminotransferase levels [4]. Between May 1986 and October 2005, 515 LT procedures were performed at our Institution in Genoa. Of these, 32 adult patients underwent first LT to treat FHF. The indication for emergency LT was made according to Kings’ College Hospital criteria [5]. During the same period, 21 patients with acute AP poisoning were treated in our dedicated intensive care unit. Six of them developed irreversible signs of poisoning, FHF and were transplanted. Fifteen patients were successfully managed without LT by intensive care medical treatment with N-acetyl cysteine infusion and high dosages of penicillin G. Furthermore, among these, three of them required MARS and two haemodiaperfusion on activate charcoal. In all these patients, the amatoxines serum level was below the lethal concentration (LC) [6]. Six LT patients had an amatoxines serum level above the LC and therefore their outcome should have been fatal without LT. The findings and course of this series of patients, which is the largest reported, was analysed and prognostic criteria defined on the basis of this experience and available data. The prognosis of these six transplanted patients was assessed by monitoring liver function tests (PTT, INR, V factor, AT III, total and split bilirubin, transaminases, WBC and platelet count, serum ammonia, blood and urinary alpha-amanitin, ascites output) plus serum creatinine level as renal failure from amatoxines is often described. AP poisoning differs from other causes of acute liver failure in several respects. The following criteria suggests how to identify a lethal from a non-lethal course: PTT <20% over the course of several days, serum creatinine concentration >1.5 mg%– raising despite careful adjustment of water and electrolyte abnormalities, serum bilirubin >5 mg %, also progressive encephalopathy and or coma indicate a lethal course [5]. In our series of transplanted patients a PTT value <20% (range: 3–17%) was observed, conversely, PTT time value >20% (range: 23–41%) was found in patients successfully treated with a conservative medical approach. The whole group of transplanted patients survived. Medium follow-up is 52 months (range: 3–122 months). LT is the procedure of choice for the treatment of acute AP poisoning complicated by FHF. The identification of correct inclusion criteria for a lethal prognosis under conservative treatment is a key point. This should be taken into account when poisoned patients are to be transferred to a transplant centre for appropriate treatment. In conclusion, based on our series of patients, we believe that the key of success is represented by many factors, including immediate organ availability for LT and careful selection of patients eligible for emergency transplantation.