Amanita Phalloides or ‘death cap’ is a cyclic peptide containing mushroom. A. Phalloides is one of the most poisonous mushrooms in the world. Cyclic peptide mushroom are involved in the majority of fatalities associated with mushroom poisoning (Enjalbert et al., 2002). Ingestion of Amanita Phalloides results in hepatotoxicity and death in severe cases. Nephrotoxicity has been also reported in clinic in patients with Amanita phalloides poisoning (Garrouste, Boudat, & Kamar, 2009). Treatment of A. Phalloides includes a wide range of supportive therapy and use of therapeutic agents mainly benzylpenicillin, silibinin and N-acetylcysteine. However, there is no clinical evidence of the efficacy of these antidotes and high mortality rates were observed even when these antidotes were administrated. Benzylpenicillin showed the highest mortality rate 11.6% in comparison to silibinin 5.4% and N-acetylcysteine 6.7% (Enjalbert et al., 2002).
Incidence of amanita phalloides intoxication have been reported mainly in Europe, but data have been published about Several cases of A. Phalloides poisoning in north-eastern United States, Central and South America, Asia, Australia and Africa (Vetter, 1998). Amanita Phalloides contains three cyclic peptides toxins phallotoxins, virotoxins and amatoxins (Vetter, 1998). Phallotoxins are bicyclic heptapeptides. Due to their insufficient absorption from the gastrointestinal track, phallotoxins are not toxic to humans. Phallotoxins are believed to cause the gastrointestinal phase clinical symptoms in patients with amanita intoxication. However, the gastrointestinal phase was recorded in a patient that consumed α-amanitin containing mushroom that did not contain phallotoxin. (Intensive & Unit, 1993). Nevertheless, recent evidence by Santi et.al. showed that phallotoxins damage the cellular membrane of the enterocytes in the intestine. Phallotoxins binds to the F-actin on the actin polymerisation depolymerisation cycle, this binding prevents microfilaments depolymerization, disturbing the correct function of the cytoskeleton resulting in impairment of the cell membrane function (Santi et al., 2012). Thereby, phallotoxins are responsible for the gastrointestinal symptoms of nausea, vomiting, and diarrhoea in A. phalloides patients. When laboratory animals were injected parenterally with phallotoxins they developed haemorrhagic necrosis of the liver (Letschert, Faulstich, Keller, & Keppler, 2006). Virotoxins are monocyclic peptides. Their structure and biological activity are somewhat similar to phallotoxins suggesting that they share a common precursor. Due to their poor oral bioavailability they have no significant toxicological effect on humans after oral ingestion.
The toxicity of A. Phalloides is attributed to amatoxins. Amatoxins are heat stable bicyclic octapeptides. They are resistant to enzyme and acid degradation, and therefore when ingested they will not be inactivated in the gastrointestinal tract. A wide variety of amatoxins have been identified and isolated however α-amanitin have been identified as the primary toxin in A. Phalloides (Magdalan et al., 2010:Santi et al., 2012).
Kinetics of α-amanitin:
α-amanitin is readily absorbed in the gastrointestinal tract, this was evident by their presence in urine within 90-120 minutes of ingestion (Einsele et al., 1987). α-amanitin does not bind to serum protein and therefore it is rapidly distributed to the liver and kidneys and eliminated from the blood (Floersheim, 1983:Flesch, Sauder, & Kopferschmitt, 1993). α-amanitin can be detected in the urine up to 3 days after ingestion (Einsele et al., 1987). The liver and kidneys are thought to be the primary target organs of the toxin and thus more likely the most affected ones. High concentrations of α-amanitin were found in the liver and kidneys however it seems that kidneys have higher concentration of the toxin (Flesch et al., 1993).Once it is distributed to the liver, α-amanitin accumulates in the hepatocytes through uptake by the Organic Anion Transport Peptide OATP. There are three OATP transporters that are located on the basolateral side of the hepatocytes OATP1B1, OATP2B1 and OATP1B3. OATP1B3 has been identified as the main transport protein that responsible for the uptake of α-amanitin inside the hepatocytes (Letschert et al., 2006). α-amanitin does not undergo metabolism and excreted unchanged in urine. α-amanitin is also excreted in the bile, Thiel et al. reported in a study using pig liver that α-amanitin was detectable in bile when the pig liver was exposed to small concentration of α-amanitin, however it was not detectable when higher concentrations were used, suggesting that α-amanitin affects the ability of the hepatocytes biliary excretion due to hepatocytes damage (Thiel et al., 2011).
Mechanism of Toxicity of amatoxins:
Several mechanisms have been attributed to the toxicity of α-amanitin. The main mechanism is the non-covalent binding to ribonucleic acid polymerase II (RNAP II) and inhibiting its activity in the nucleus (Santi et al., 2012). It has been proposed that α-amanitin inhibits RNAP II by direct interference with the trigger loop, therefore preventing the conformational change of RNAP II and inhibiting the ribonucleic acid (RNA) elongation process. A recent in silico study showed that the α-amanitin site is beneath a ‘‘bridge helix’’ extending across the cleft between the two largest polymerase II subunits, Rpb1 and Rpb2. This binding of α-amanitin constrain the movement of the bridge helix which is required for the translocation of DNA and RNA need for the synthesis. α-amanitin does not affect the affinity of Polymerase II for nucleotides. However it prevents the translocation needed to empty the site for the next round of synthesis (Bushnell, Cramer, & Kornberg, 2002). Inhibition of messenger RNA (mRNA) synthesis results in decline in protein synthesis which eventually lead to cell death (Santi et al., 2012). However, in vitro and in vivo studies suggested that is α-amanitin also causes apoptotic cell death. In vitro exposure of human hepatocytes to α-amanitin resulted in induction of p53 and caspase 3 dependant apoptosis. In vivo Knockout p53 mice showed marked resistance towards α-amanitin induced liver damage, while wild-type mice in the same conditions underwent liver damage (Garcia et al., 2015).
Other mechanisms might be involved in α-amanitin-induced toxicity. Leist et al. observed in a study using in vivo and in vitro. In vivo using human hepatoma cell TNF messenger RNA levels increased and hepatocytes underwent apoptosis. Whereas liver injury caused by α-amanitin in mice after being treated with anti TNF antibodies was prevented. In addition, transgenic mice lacking TNF-α receptor seem to be relatively resistant to α-amanitin-induced toxicity. Therefore, hepatocyte apoptosis may result from a synergistic action between α-amanitin and TNF-α (Leist et al., 1997). However, the mechanisms of such synergistic effects remain unclear and the dependence of α-amanitin toxicity on the presence of TNF-α was not confirmed in another study using rat hepatocyte cultures. Thus, TNF-α may not be involved in the development of cytotoxicity by α-amanitin but exacerbates it (El-Bahay et al., 1999). In addition, the hepatic accumulation of α-amanitin leads to an increase of superoxide dismutase. However, investigations are still needed to completely clarify the pathophysiology of ROS in the α-amanitin- induced hepatotoxicity (Garcia et al., 2015).
α-amanitin nephrotoxicity has been reported in the clinic (Garrouste et al., 2009). It is believed that since α-amanitin is primary eliminated through the kidneys, it would accumulate in the kidney and induces renal function impairment through interacting with RNA polymerase II in the kidney cells (Kirchmair et al., 2012:Flesch et al., 1993). Histological investigation of renal tissue collected from rabbit and human after exposure to α-amanitin, revelled the presence of lesions in all parts of the nephron and renal damage were observed (Fineschi, Di Paolo, & Centini, 1996). However, it still remains unknown whether this nephrotoxicity is due to direct effect of α-amanitin or an indirect effect through the induced hepatotoxicity (Kirchmair et al., 2012:Mengs, Torsten Pohl, & Mitchell, 2012:G Vogel, Braatz, & Mengs, 1979).
The symptoms of A. Phalloides intoxication become evident after few hours after ingestion of the mushroom. The symptoms can vary from simple gastrointestinal disturbances to severe liver damage leading to death in serve cases. The severity of A. phalloides intoxication depends on the amount of toxin ingested and the time delay between ingestion and initiation of treatment (Broussard et al., 2001). There are three distinct phases of A. Phalloides toxic syndrome have been established in literature: 1) Gastrointestinal phase, 2) Latent phase and 3) The hepatorenal phase (Garcia et al., 2015). Table (1) provides a short description of the stages of A. phalloides intoxication and their symptoms.
There are no apparent symptoms after the ingestion of A. Phalloides instead there is asymptomatic period for 6-24 hours before the onset of the gastrointestinal phase. The gastrointestinal phase is characterized by onset of severe gastrointestinal disturbances. These symptoms include vomiting, severe watery diarrhea similar to cholera and abdominal pain. The symptoms normally last for 1-2 days, if not treated properly the patient may in this phase get severely dehydrated and hypovolemic with subsequent circulatory instability, oliguria and a functional renal insufficiency. Metabolic disorders like hypoglycaemia, electrolyte disturbances, hypokalaemia and metabolic acidosis may also develop. Other symptoms might also develop during this phase tachycardia, hypoglycaemia, dehydration, and electrolyte imbalance. It has been suggested that the presence of phallotoxins in theses mushroom results in this phase. Phallotoxins damages the enterocytes cell membrane (Broussard et al., 2001).
The second phase is the latent phase which is characterized by absence of symptoms, whilst progressive deterioration of hepatic and renal function is occurring. The hepatic deterioration is observed in serum by the increased concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). The blood coagulation is also severely disturbed, which may give rise to internal bleeding. The pathological hallmark of amatoxin poisoning is the development of liver necrosis and this characterizes the hepatorenal phase. The patients progressively lose kidney and liver functions and may develop jaundice, hypoglycaemia, oliguria, delirium, and confusion. This phase culminates in rapid deterioration of central nervous system, severe haemorrhagic manifestations, renal and hepatic failure, which corresponds to a bad prognosis. About 20–79% of the intoxicated patients develop chronic liver disease(Garcia et al., 2015).
Table (1): The onset and the clinical symptoms of amanita intoxication phases:
Onset from ingestion Clinical symptoms
6-24 hours vomiting, crampy abdominal pain, and severe secretory diarrhoea
24-72 hours Asymptomatic, worsening of hepatic and renal function
4-9 days deterioration of central nervous system, haemorrhagic manifestations, renal and hepatic failure
There is no worldwide accepted guidelines for treatment of amanitin intoxication (Garcia et al., 2015). All treatment measures taken focuses on stabilization of vital function, reducing the absorption of the toxin, increasing the elimination of amatoxins and the use of chemotherapeutic agents. Some of these measures have some degree of success while mostly show small efficacy. Supportive measures are taken for the management of gastroenteritis and hepatotoxicity. In the gastrointestinal phase, hypovolemic shock could be managed by the administration of intravenous ﬂuids to restore the electrolyte abnormalities, metabolic acidosis and hypoglycaemia. Due to the enterohepatic circulation of amatoxins it was thought that administration of activated charcoal could reduce amatoxins absorption in the gastrointestinal track. However, there is no evidence that administration of activated charcoal would improve the clinical outcome. Other measurement taken for treatment of hepatoxicity include correction of coagulation disorders by parenteral vitamin K (10mg daily for three consecutive days) and fresh frozen plasma and antithrombin III. Oral lactulose and neomycin to prevent encephalopathy and mannitol to lower intracranial pressure and avoid cerebral oedema (Enjalbert et al., 2002).
α-amanitin is detectable in blood with in 24 to 48 hours post ingestion. For that reason, removing amatoxins by haemodialysis, hemoperfusion or plasmapheresis was considered to have no impact on patient survival. Since α-amanitin is excreted mostly in urine it was believed that forced diuresis would potentially increase the renal clearance and could be a potential good therapy for this intoxication. However there is no evidence that this method would decrease the amount of α-amanitin in the liver (Flesch et al., 1993).
There is no specific antidote for amanita intoxication however several therapeutics agents such as benzylpenicillin, silibinin and N-acetylcysteine are used as antidotes either alone or in combination. There are many different opinions in reference databases on the choice of treatment. The toxicology database of the national poisons information service (TOXBASE) in the United Kingdom recommends the use of penicillin G, whereas the national poisoning center of New Zealand recommends the use of silibinin. The Portuguese poisoning center (CIAV) include both antidotes as suitable for the treatment of this intoxication. The Dutch toxicology centre recommends the usage of silibinin as the main antidotes against A. phalloides, in the past benzylpenicillin was used. However, it has become clear that benzylpenicillin does not contribute to an improved outcome of the patient. The use of these antidotes is still associated with high mortality rates table. Moreover, there is no clinical evidence of the efficacy of these antidotes.
Investigations using isolated rat hepatocytes showed that amanita extract decreased the intracellular glutathione content of the hepatocytes. This clinical similarity between acetaminophen overdose and α-amanitin poisoning suggested that N-acetylcysteine could be included in the management of A. Phalloides intoxication. NAC is a glutathione precursor when natural stores are depleted and a free radical scavenger. There is no enough clinical data about the efficacy of NAC as an antidote in amanita intoxication, therefore some protocols exclude NAC from the therapy. When NAC is used, the recommended dose is 150 mg/kg intravenously over 15 minutes as a loading dose followed by 50 mg/kg over 4 hours followed by 100 mg/kg over 16 hours. This dosing scheme is the same for the intoxication of acetaminophen. NAC is used as a protective substance and almost never used as monotherapy. A retrospective study was performed to compare the overall survival in patients with and without additional treatment with NAC. This study showed a lower mortality rate for the patient group treated with NAC (Akın, Keşkek, Kılıç, Aliustaoğlu, & Keşkek, 2013).
Is the most commonly used antidote either in monotherapy or in combination. In 2002 Enjalbert et.al, has documented in his survey that over the past 20-year period, benzylpenicillin was the most used antidote in management of amanita intoxication 1411 out of 1632 of the treated patients (86.5%) received benzylpenicillin (Enjalbert et al., 2002). Benzylpenicillin is administrated either as mono-chemotherapy or combined with other drugs. It was believed that benzylpenicillin has a hepatoprotective effect in α-amanitin poisoning. In vitro studies and animal studies using mice and rat showed that benzylpenicillin prevented both the rise of liver enzymes and the fall of clotting factors in plasma. Several theories were proposed to explain the protective effect of benzylpenicillin in α-amanitin poisoning. It was suggested that benzylpenicillin displaces α-amanitin from its binding site in serum albumin. However , Floersheim et.al studied this hypothesis and failed to detect any binding of α-amanitin to serum protein (Floersheim, 1983). Recent in-vitro studies using human hepatocytes suggested that benzylpenicillin blocks the uptake of α-amanitin by inhibition of organic anion transport 0ATP3 (Letschert, Faulstich, Keller, & Keppler, 2006).
Despite the reported benzylpenicillin efficacy, it has some major safety issues, administration of benzylpenicillin commonly cases allergic drug reaction in 1-10% of the patients. The high amount of sodium administrated with benzylpenicillin – The recommended dose of benzylpenicillin is 1million IU/kg/day in treatment of patients intoxicated with of α-amanitin may result in disruption of electrolyte balance and severe granulocytopenia. In addition, it may evoke neurotoxic symptoms in patients with nervous system disease and renal insufficiency (Enjalbert et al., 2002).
The recommended dose of benzylpenicillin is 1million IU/kg/day according the Portuguese poisoning information centre. TOXBASE recommends a dose of 0.5 million units/kg/day as a continuous infusion for 2–3 days after the day of ingestion, with close monitoring of renal function. However, the Dutch and New Zealand national poisoning centre doesn’t recommend the use of benzylpenicillin due to its safety issues (Garcia et al., 2015).
Silibinin Is a hepatoprotective flavonolignan extracted from the milk thistle plant Silybum marianum. Due to its antioxidant activity silibinin was used in management of α-amanitin poisoning. The effects of silibinin were reported by Han et.al. Silibinin increased the survival rate of mice injected intraperitoneally IP with A. Phalloides extract. The efficacy of silibinin depends on the time of administration after ingestion of the mushroom and the severity of the symptoms (Enjalbert et al., 2002). Animal studies using dogs showed that silibinin reduced the extent of liver damage after oral A. phalloides extract (Günther Vogel, Tuchweber, Trost, & Mengs, 1984).
Silibinin main mechanism of action is inhibiting the uptake and accumulation of α-amanitin in the hepatocytes by competitive binding to OATP3 transporter. It also acts as a radical scavenger, silibinin reduces the free radical load and stimulate the activity of SOD and increase GSH levels (Fraschini, Demartini, & Esposti, 2002). Moreover, in vivo and in vitro studies showed that silibinin stimulates ribosomal formation, DNA and protein synthesis. Silibinin protein synthesis, Silibinin binds to RNA polymerase I at specific binding sites increasing the transcription of ribosomal RNA, which may counterbalance the inhibition of RNA polymerase II induced by amatoxins (Fraschini, Demartini, & Esposti, 2002:Pradhan & Girish, 2013). Based on animal studies and limited human data, it seems that silibinin is the most promising molecule to prevent pathophysiological events after amatoxin intoxications, with a good safety profile. Therefore, CIAV, the national poisons centre of New Zealand and the Dutch toxicology center recommend an intravenous administration of 20– 50 mg/kg/day in four divided doses. Treatment should be continued for 48-96h after mushroom ingestion. TOXBASE recommendations for A. phalloides poisoning treatment do not include silibinin administration probably due to the low clinical evidence available so far concerning silibinin efficacy (Garcia et al., 2015).
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