Gyromitrin (see Figure 2(Fig. 2)) has the chemical formula C₄H₈N₂O and is named N'-ethylidene-N-methylformohydrazide (Horowitz and Horowitz, 2017[53]). Since gyromitrin has a boiling point of 143 ºC (289 ºF) and is water-soluble and volatile, a thorough boiling or long-term drying is necessary to partially reduce the toxicity of gyromitrin-containing mushrooms (Michelot and Toth, 1991[72]). Gyromitrin when consumed is converted into toxic metabolites N-methyl-N-formylhydrazine (MFH) and N-methylhydrazine (MMH).

Acute oral toxicity (LD₅₀) of gyromitrin is markedly variable in different animal models: 344 mg/kg in mice (Wright et al., 1978[119]), and 320 mg/kg in rats (Mäkinen et al., 1977[69]), whereas in rabbits it varies from 50 mg/kg (Pyysalo, 1975[85]) to 70 mg/kg (Mäkinen et al., 1977[69]). Chickens, otherwise, seem to be insensitive to gyromitrin intoxication since no toxic effects were detected with doses of 400 mg/kg (Mäkinen et al., 1977[69]). The rabbits' and rats' poisoning symptoms included convulsions, hypersensitivity, loss of activity, lack of appetite, and severe weight loss intoxication. Rabbit's necropsy showed extensive fatty degeneration of the liver, but this effect was much less severe in rats. The human LD₅₀ of gyromitrin has been evaluated as 20-50 mg/kg for adults and 10-30 mg/kg for children. These doses correspond approximately to 0.4-1 kg and 0.2-0.6 kg of fresh G. esculenta (Michelot and Toth, 1991[72]).

The toxicokinetics of gyromitrin data on biotransformation are scarce. Toxicosis can result from oral and inhalation exposure. Ingestion of gyromitrin (acetaldehyde N-methyl-N-formylhydrazone)-containing mushrooms results in the hydrolysis of gyromitrin to MFH, which is further metabolized to MMH. The degree of hydrolysis is dependent on the pH in the stomach, but it is not complete (Wright et al., 1978[117]). Inhaling vapors during the cooking process can also result in poisoning. Hydrazines are further converted in the liver to reactive intermediates such as methyl cations and free methyl radicals.

Once ingested, gyromitrin is rapidly hydrolyzed in the stomach (acidic conditions), forming easily absorbed hydrazines MFH and MMH (Michelot and Toth, 1991[72]). Whereas the exact mechanism of toxicity remains unknown, several studies have demonstrated many effects in multiple organ systems. The oxidation of MFH was investigated to determine the intermediates that may develop during metabolism (Gannett et al., 1991[38]). MFH oxidation yielded formaldehyde and acetaldehyde. The formation of acetaldehyde requires the oxidation of MFH to a diazenium ion (I) or diazene (II) and fragmentation of I/II to formyl and methyl radicals (Figure 3(Fig. 3)) (Gannett et al., 1991[38]).

Moreover, MFH is a non-competitive inhibitor of human intestinal diamine oxidase (ID₅₀ = 1.6 x10^-5 mol/L), this concentration corresponding to less than 5 g of fresh mushroom. Noteworthy, strongly decreased intestinal diamine oxidase (DAO) activity has been linked to intestinal disorders with the proliferation of the mucosa (Biegński et al., 1984[16]).

Another enzyme that is directly inhibited by gyromitrin is the pyridoxal phosphokinase. This enzyme is responsible for dietary vitamin B6 (pyridoxine) conversion into active pyridoxal 5-phosphate (Horowitz et al., 2024[53]). Moreover, in vitro and in vivo, MMH may generate hydrazones with pyridoxal-5-phosphate (Barceloux, 2008[8]). Pyridoxal-5-phosphate is a cofactor for glutamic acid decarboxylase and GABA transaminase in the gamma-aminobutyric acid (GABA) synthetic pathway, which results in decreased GABA synthesis (Barceloux, 2008[8]). MMH can directly block glutamic acid decarboxylase when given intraperitoneally to rats at a concentration of 0.8 mM/kg, resulting in a further decrease in GABA levels (Medina 1963[71]). Hydrazones may also cause lipid peroxidation in the liver, leading to cytotoxicity and acute liver damage. Hydrazones might potentially act as oxidizing agents, causing methemoglobinemia. Although methemoglobin production is frequently discussed as a potentially harmful consequence of gyromitrin mushroom ingestion in humans, there is just one case record in a dog (Horowitz et al., 2024[53]).

MMH toxicity has also been linked to the formation of free radical intermediates that produce glutathione depletion in isolated hepatocytes and liver microsomes (Albano et al., 1989[1]).

MMH, (Toth and Shimizu 1973[110]) as well as its precursors MFH (Toth and Patil, 1979[106], 1982[107]; Toth et al., 1979[108]) and raw G. esculenta (Toth et al., 1992[109]), have been shown to be carcinogenic in in vivo studies. G. esculenta, when administered orally to Swiss mice (6 weeks), could induce tumors in the lungs, nasal cavity, blood vessels, forestomach, glandular stomach, cecum, and liver (Toth et al., 1992[109]).

The MMH administration of 0.01 % solution in the drinking water to 6-week-old Syrian golden hamsters (lifetime) was able to generate malignant histiocytomas of the liver and cecum tumors (Toth and Shimizu, 1973[110]). In mice, MFH has also been shown to be tumorigenic to several organs (Toth and Patil, 1982[107]; Toth et al., 1979[108]). Similarly, MFH administration in drinking water as a 0.0039 % solution to randomly bred Swiss albino mice (six weeks old) throughout life was able to induce tumors in the lungs, livers, blood vessels, gall bladder, and bile ducts. This effect was also observed in Syrian golden hamsters, with MFH causing the formation of benign and malignant liver cell tumors, malignant histiocytomas, and tumors of the gallbladder and bile ducts after administration of 0.0078 % MFH in drinking water (Toth and Patil, 1979[106]). MFH was also shown to be mutagenic in the AMES assay, especially in the presence of a metabolic activation system (Von Der Hude and Braun, 1983[114]). It is suggested that the oxidation of MFH to a diazenium ion (I) or diazene (II) intermediates may be important in understanding and elucidating carcinogenesis by MFH (Gannett et al., 1991[38]). However, in humans, no carcinogenic effects were reported since there are no case reports and/or epidemiological studies in the literature, which is probably due to the sporadic consumption of this mushroom.

Initial clinical symptoms are associated with gastrointestinal (GI) and neurological disorders. Nausea, vomiting, headache, abdominal pain, and occasionally watery or bloody diarrhea and hypoglycemia may occur after a latency period of 6-8 hours. In most cases, the poisonings are limited to gastrointestinal disorders, and the patient recovers 2 or 5 days later without any after effects (Michelot and Toth, 1991[72]). In severe cases of intoxication, neurological disorders, such as nervousness, delirium, coma, and convulsions, complete the clinical condition (Michelot and Toth, 1991[72]). It may, rarely, occur epileptogenic neurotoxicity, with vertigo, delirium, seizures, stupor, and coma, especially in the elderly and in patients on isoniazid prophylaxis or treatment for tuberculosis (Diaz, 2005[28]). Hepatic and renal damage are potential complications of gyromitrin poisoning and may be fatal in 2-4 % of cases (Barceloux, 2008[8]; Singh et al., 2019[99]).

In the literature, only a few quantitative analytical methodologies for gyromitrin have been published. Older methods for the evaluation of gyromitrin in mushrooms were based on thin-layer chromatography (TLC) and spectrofluorometry (gyromitrin and MMH) (Andary et al., 1984[3]). However, most of the studies use gas chromatography (GC-MS) to determine gyromitrin and MMH levels in mushrooms. The method consists of determining the total hydrazones content based on acid hydrolysis of gyromitrin and other related hydrazones in air-dried Gyromitra esculenta followed by derivatization of MMH with pentafluorobenzoyl chloride and subsequent determination by GC-MS (Arshadi et al., 2006[6]). MMH was detected in mice peritoneal fluid by gas-liquid chromatography (Von Wright et al., 1978[115]) and in rat serum by high-performance liquid chromatography (HPLC) with electrochemical detection (Slanina et al., 1993[100]). Gyromitrin was determined using glass-capillary gas chromatography and NMR spectroscopy (Mäkinen et al., 1977[69]) and there is a report of post-mortem examinations of gyromitrin in viscera by IR and UV spectroscopy and TLC (Giusti and Carnevale, 1974[46]). More recently, a theoretical description has been performed for the determination of gyromitrin through an electrochemical methodology. In this view, Tkach et al., (2019[104]) concluded that vanadium (III) oxyhydroxide may be efficient in the determination of gyromitrin. For that, the electroanalytical process is performed cathodically with a gradual reduction of the products, becoming an efficient and diffusion-controlled method for gyromitrin determination (Tkach et al., 2019[104]).

The recognition of gyromitrin-containing mushrooms in meals aids in the diagnosis of gyromitrin toxicosis. To distinguish between the authentic and the fake morels, a mycologist's identification is required. Because detecting gyromitrin, hydrazine analogs, or metabolites in mushrooms or biological materials is not regularly accessible, diagnosis is based mostly on clinical and clinicopathological symptoms, as well as mushroom identification (Puschner, 2018[83]).

After exposure to gyromitrin, most people only have minor gastrointestinal problems and recover completely within a few days. As of the late onset of clinical symptoms, early decontamination is sometimes impossible. Activated charcoal has been advocated for treatment, despite the lack of effectiveness evidence (Karlson-Stiber and Persson, 2003[57]). In cases of severe central nervous system toxicity, it is relevant to give pyridoxine (25 mg/kg or 5 g intravenous dose up to a daily adult dose of 15 - 20 g) since it counteracts the inhibition of GABA synthesis (Barceloux, 2008[8]). The administration of benzodiazepines, such as lorazepam is also used in case of seizures. Administration of folinic acid and N-acetylcysteine has been recommended in humans, however, controlled studies have not been performed, and thus their relevance remains to be established (Puschner, 2012[84]).

The content of orellanine was determined in dried fungus and the amounts range from 9 mg/g in C. speciosissimus to 14 mg/g in C. orellanus (Karlson-Stiber and Persson, 2003[57]). There are significant inter- and intra-species differences in the toxic reaction to orellanine, according to the literature. In mice, the intraperitoneal and oral LD₅₀ are respectively 12.5 and 90 mg/kg (Richard et al., 1988[89]), however, in another study the intraperitoneal and oral LD₅₀ are 15 and 33 mg/kg, respectively (Prast et al., 1988[82]). Oral administration of C. orellanus mushrooms to male Sprague Dawley rats resulted in a LD₅₀ of 967 mg/kg body weight. The gender also appears to influence the toxicity since rat females seem to be more resistant to oral orellanine (Nieminen and Pyy, 1976[75]). Likewise, in humans, the inter-individual variability is also confirmed (Danel et al., 2001[25]). The human lethal dose of fresh mushrooms was estimated to be 100 - 200 g (Grzymala, 1957[48]). According to animal research, a 70-kg person's deadly dosage would be about 7.5 mg/kg of body weight, which would be found in roughly 70 g of dry mushrooms or 700 g of fresh mushrooms (Raff et al., 1992[86]).

The toxicokinetics of orellanine in humans are not fully understood since samples are not obtained until several days after ingestion due to the long latent period before the onset of illness (Dinis-Oliveira et al., 2016[30]). After oral administration of 2 g of C. orellanus to male Sprague Dawley rats, detectable levels of orellanine were excreted only during the first 24 hours (Prast et al., 1988[82]). In another in vivo study using rats, orellanine was almost exclusively eliminated by glomerular filtration as well as by peritoneal dialysis (Najar et al., 2018[74]). In human cases of orellanine poisoning, the toxin was not detected in urine samples collected from 2 to 18 days after ingestion (Dinis-Oliveira et al., 2016[30]). According to Rohrmoser et al. (1997[92]), orellanine and its decomposition products cannot be detected in urine, plasma, and dialysis fluids after the onset of symptoms, as most of the toxin is concentrated in the kidneys at this time (Rohrmoser et al., 1997[92]). Orellanine was qualitatively detectable in small quantities of renal biopsy samples taken 9 and 60 days after the mushroom ingestion, which suggests a relatively long period after the onset of intoxication (Rohrmoser et al., 1997[92]).

The toxicity mechanism of orellanine is not yet fully understood. Orellanine is reported to be converted to orellinine, which is then reduced to harmless orelline via photochemical degradation (Figure 4(Fig. 4)). Orellanine's chemical structure is similar to that of the pyridine herbicides diquat (1,1'-ethylene-2,2'-bipyridinium) and paraquat (1,1'-dimethyl-4,4'-bipyridinium), leading some researchers to propose a monoelectronic reduction process (generation of a stable radical) (Cantin et al., 1988[22]; Richard et al., 1987[90]). However, an in vivo analysis of orellanine's electrochemical behavior indicates that its mode of action must be distinct from that of other poisons. However, because orellanine has a substantially higher negative redox potential than paraquat and diquat, this hypothesis has been questioned. Orellanine disrupts LLC-PK1 cell monolayers and inhibits membrane-bound alkaline phosphatase and cytosolic lactate dehydrogenase activity. Orellanine's intracellular mechanism of action is confirmed by the fact that the cell membrane remains undamaged even after the monolayer disruption has begun (Heufler et al., 1987[51]). When LLC-PK1 monolayer and Caco-2 cell cultures were treated with orellanine a complete inhibition of alkaline phosphatase activity could be observed after 24 hours of incubation with 1 mM orellanine (Ruedl et al., 1989[94]). Kinetic studies were performed with renal, intestinal, and placental alkaline phosphatase isoenzymes using different cell homogenates to investigate the specific inhibitory actions of orellanine. Orellanine acts on renal alkaline phosphatase of LLC-PK1 cells as well as on bovine kidney preparation as a non-competitive inhibitor and as a competitive inhibitor on intestinal alkaline phosphatases from Caco-2 cell and bovine intestinal mucosa preparation as well as placental isoenzyme (Ruedl et al., 1989[94]). A study using the electron spin resonance (ESR) technique supported the participation of radicals in the toxicity mechanism (Richard et al., 1995[88]). Richard et al. (1995[88]) observed the formation of a radical form of orellanine identified as ortho-semiquinone anion radical which was generated by oxidation of orellanine through a variety of methods, including photooxidation (visible and UV oxidation), chemical [hydrogen peroxide (H₂O₂)], biochemical (e.g cytochrome C), and enzymatic oxidation (mushroom tyrosinase-O₂ system) (Richard et al., 1995[88]). The ortho-semiquinone anion radical is responsible for superoxide hydrogen peroxide, and hydroxyl radicals production which reduces glutathione (GSH). Thereby, oxidative stress seems to be the main orellanine toxic mechanism which may lead to large oxygen consumption, creating hypoxic conditions, as well as depletion of renal glutathione level (Richard et al., 1995[88]). Corroborating this hypothesis, ESR and spin-trapping studies, demonstrate that orellanine can be oxidized to an ortho-semiquinone radical intermediate by horseradish peroxidase (HRP) and the H₂O₂ system (Oubrahim et al., 1998[78]). Furthermore, the production of ascorbyl or glutathionyl radicals was seen when orellanine was oxidized by HRP/ H₂O₂ in the presence of reducing agents of biological importance such as ascorbic acid or glutathione (Oubrahim et al., 1998[78]). To support these findings an in vivo study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, received various doses of intraperitoneal doses of orellanine (0-5 mg/kg). One week later, renal function (GFR), ascorbyl radicals, oxidative protein damage, renal mRNA levels, catalase, glutathione peroxidase, and superoxide dismutase were evaluated. It was evident that orellanine caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. In addition, the expression of mRNA for antioxidative enzyme-related genes was strongly decreased. These data strongly suggest that orellanine nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes (Nilsson et al., 2008[76]).

Orellanine poisoning is characterized by a long latency period (2-6 days) before the onset of the disease. It follows a prerenal gastrointestinal phase, which is characterized by vomiting, nausea, and diarrhea. Other signs may coexist such as asthenia, lumbar pain, anorexia, thirst, chills, headache, and myalgia. Clinical signs may spontaneously disappear in some patients, leaving the poisoning unnoticed; in others, the signs become more intense and are accompanied by neurological manifestations (paraesthesia, taste impairment, cognitive disorders), lumbar pain, and anuria requiring hospitalization (Bouget et al., 1990[18]). The hallmark of orellanine toxicity is acute renal failure which is accompanied by several renal damage symptoms such as lumbar and flank pains, intense thirst, oliguria, and polyuria (Danel et al., 2001[25]; Dinis-Oliveira et al., 2016[30]). Early and severe interstitial fibrosis, interstitial edema, and tubular epithelial necrosis are the most common features of renal lesions (Duvic et al., 2003[31]). The incidence of acute renal failure varies from 30 to 46 % and it depends on individual susceptibility, pre-existing renal disease, and the cumulative dose of toxin ingested (Duvic et al., 2003[31]), and in 50 % of the cases may progress to chronic renal insufficiency (Flesch and Saviuc; 2004[36]). Moreover, renal failure occurs 4 - 15 days (median: 8 days) after ingestion depending on the dose ingested (Barceloux; 2008[8]). A renal biopsy performed on two patients showed pronounced focal tubular damage with tubulorrhexis, cast formation, and severe interstitial edema with patchy infiltration of lymphocytes, plasma cells, and some polymorphs (Short et al., 1980[98]). Another kidney biopsy obtained from 5 patients performed 10-21 days after fungal ingestion showed a similar pattern dominated by acute tubular necrosis, tubular dilatation, tubular cellular atrophy and degeneration, apoptotic bodies, interstitial edema, and focal inflammation but no eosinophilia (Hedman et al., 2017[49]). Liver injury has also been shown based on the analysis of transaminases and bilirubin levels, as well as hepatomegaly, hepatalgia, and necrosis lesions, nevertheless, most of the studies ruled out liver involvement (Dinis-Oliveira et al., 2016[30]).

Orellanine has been isolated and analyzed using a variety of analytical methods. A color test based on the ferric-orellanine reaction has been developed for routine identification of the toxin in mushroom samples. A fresh or dried mushroom is crushed in five volumes of water and filtered after 10 min at room temperature. The filtrate is then mixed with an equal amount of 3 % ferric chloride hexahydrate dissolved in 0.5 N hydrochloric acid (HCl). The presence of orellanine is suspected if a dark gray-blue color ink appears (Dinis-Oliveira et al., 2016[30]). Quickly and simple analytical methods are available to detect orellanine using TLC on silica-gel or cellulose plates which allow for analysis of the toxin in mushroom or tissue samples (Rapior et al., 1989[87]; Rohrmoser et al., 1997[92]). Other analytical procedures are available such as electron spin resonance spectroscopy (ESR) analysis which allowed the detection of the oxidation product of orellanine (Oubrahim et al., 1997[79]). HPLC methods with electrochemical (Holmdahl et al., 1987[52]), UV (Cantin et al., 1989[21]), and diode-array (Koller et al., 2002[60]) detectors have also been described. These HPLC methods were developed to analyze orellanine in mice serum directly (Holmdahl et al., 1987[52]) and mushrooms (Cantin et al., 1989[21]; Koller et al., 2002[60]). Additionally, an HPLC-electrospray tandem mass spectrometry method for the analysis of total orellanine in mushrooms and plasma is also available in the literature (Herrmann et al., 2012[50]). Moreover, to analyze orellanine in tissues, HPLC and LC-MS/MS methods were developed (Srivastava et al., 2016[101]).

Alternatively, a more sensitive GC-MS method can be used for the analysis of orellanine in biological samples (Brondz et al., 2012[20]). The direct identification of orellanine in the stomach contents of rats after they were fed food containing C. orellanus was achieved using GC-MS with supersonic molecular beams (SMB) (Brondz et al., 2012[20]). According to the authors, the GC approach gives much greater separation of compounds in a combination than TLC or HPLC, as well as higher repeatability (Brondz et al., 2012[20]).

Unfortunately, so far, there is no known antidote against orellanine poisoning. Principles of treatment include limiting absorption, enhancing elimination, correcting metabolic abnormalities, and providing supportive care (Barceloux, 2008[8]; Danel et al., 2001[25]; Dinis-Oliveira et al., 2016[30]). The decontamination of poisoned patients using emesis or gastric lavage is useful if the patients seek medical care earlier than 6 hours after ingestion (Dinis-Oliveira et al., 2016[30]). Early secondary decontamination by either plasmapheresis or hemoperfusion has no proven benefit in removing orellanine and preventing renal failure after Cortinarius ingestions (Diaz, 2005[28]). As the kidneys seem to be the target organ of orellanine poisoning a periodic accurate assessment of changes in renal function is crucial for monitoring the renal function. At the late stage of severe chronic renal insufficiency, the renal transplant is a last resort treatment measure (Danel et al., 2001[25]). Renal transplantation should not be performed too early in the course of illness, as complete renal recovery may occur in 40-60 % of Cortinarius-poisoned patients (Diaz, 2005[28]). As the orellanine mechanism of toxicity seems to involve reactive oxygen species with large oxygen consumption and depletion of glutathione some authors recommend the use of N-acetylcysteine, corticosteroids, and selenium (Kilner et al., 1999[59]; Wörnle et al., 2004[118]). In fact, Kerschbaum et al. (2012[58]) reported a case of a couple with acute renal failure after accidental intake of C. rubellus and a favorable outcome after treatment with high-dose antioxidant therapy with N-acetylcysteine and steroids. Thereby, the authors suggested that early treatment with antioxidant therapy and steroids might be effective in reducing the risk of developing end-stage renal failure (Kerschbaum et al., 2012[58]). However more studies are needed to strengthen this conclusion.

Phallotoxins are bicyclic heptapeptides that include phalloidin, phalloin, prophallin, phallisin, phallicin, phallacidin, and phallisacin (Figure 5(Fig. 5)) (Vetter, 1998[112]). Despite phallotoxin's high toxicity, these toxins are not able to be absorbed by the gastrointestinal tract, suggesting negligible effect in human poisoning. Therefore, the toxicity of cyclopeptide mushrooms is mostly due to the amatoxins, which will be the focus of the discussion in the following sections (Karlson-Stiber and Persson, 2003[57]).

Amatoxins are bicyclic octapeptides and consist of nine defined members: α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amanin amide, amanullin, amanullinic acid, proamanullin (Vetter 1998[112]). From these, alpha and beta-amanitins predominate producing systemic toxicity (Schneider 2014[95]). Specific properties characterized these toxins: heat stability, water solubility, and resistance to enzyme degradation (Poucheret et al., 2010[80]). All groups of toxic peptides contain a tryptophan residue substituted at position 2 of the indole ring by a sulfur atom (Figure 6(Fig. 6)).

Only the number of hydroxy groups and an amide carboxy exchange distinguish amatoxins. This group of toxins can be found in the Amanita, Lepiota, and Galerina species (e.g. A. phalloides, A. virosa, A. verna, A. ocreata, A. bisporigera, A. suballiacea, A. tenuifolia, and A. hygroscopica) (Karlson-Stiber and Persson, 2003[57]). From these, A. phalloides are responsible for the majority of fatalities caused by mushroom poisoning (Garcia et al., 2015[42]). It is the most toxic species which, apart from rare exceptions, contains 2 to 3 mg of amatoxins per gram of dry tissue. This mushroom is found growing all over central Europe and can easily be confused with the edible Horse Mushroom (Agaricus arvensis) and Tricholoma equestre. At an early development stage, an Amanita “button” can easily be mistaken for an edible puffball (Lycoperdon bovista) (Garcia et al., 2015[42]).

As monotherapy or in combination with other drugs, benzylpenicillin (Figure 8(Fig. 8)) and other β-lactam antibiotics have been the most commonly used drugs in the treatment of amatoxin poisoning. However, the US Food and Drug Administration (FDA) has not authorized benzylpenicillin for the treatment of amanita poisoning (Enjalbert et al., 2002[33]).

The ability of benzylpenicillin to block amatoxin from entering hepatocytes and to directly compete with it for transmembrane transport has previously been proven (Letschert et al., 2006[64]).

Tong et al. (2007[105]) compared different treatments, including benzylpenicillin, to reduce liver damage in a mouse model of amatoxin-induced liver toxicity. They injected 0.6 mg/kg a-amanitin into six groups of mice over 48 hours. After 4 hours, the mice were randomly assigned to receive one of five intraperitoneal treatments (N-acetylcysteine, benzylpenicillin, cimetidine, thioctic acid, or silybin). These treatments were repeated every 4 to 6 hours. After 48 hours, the mice were sacrificed, and their liver enzyme levels (alanine transaminase and aspartate transaminase) were measured from aspirated cardiac blood. Liver samples were also collected to assess the extent of liver damage. The results suggested that benzylpenicillin was not effective in limiting hepatic injury after a-amanitin poisoning. Increases in aminotransferases and hepatonecrosis were not attenuated by this antidote (Tong et al., 2007[105]).

A therapeutic approach combining benzylpenicillin infusions with traditional support measures was administered to 33 severe A. phalloides poisoning cases in humans, resulting in a 100 % survival rate. Moroni et al. (1976[73]) consider benzylpenicillin a novel method capable of entirely altering the outcome of this mushroom poisoning when used appropriately alongside standard treatment (Moroni et al., 1976[73]).

Magdalan et al., (2010[67]) studied the effects of benzylpenicillin, N-acetylcysteine, and silybin as antidotes in human hepatocytes exposed to α-amanitin. They cultured primary hepatocytes for 48 hours with daily doses of α-amanitin (2 μM) and/or different concentrations of antidotes. Cell cytotoxicity assessment was conducted at 12, 24, and 36 hours of exposure to α-amanitin and/or antidotes. The findings demonstrated that N-acetylcysteine, benzylpenicillin, and silybin incubation in human hepatocyte cultures exhibited a similarly robust protective effect against α-amanitin-induced cell damage (Magdalan et al., 2010[67]).

In another study, benzylpenicillin's effectiveness was compared to ceftazidime and rifamycin SV in a human hepatocyte model. Primary hepatocyte cultures were maintained for 48 hours with daily doses of α-amanitin (2 μM) and/or the tested antidotes. Cytotoxicity assessment of the cultured cells was conducted after 48 hours of exposure to α-amanitin. The study's results provide proof that co-incubation of α-amanitin and benzylpenicillin effectively shielded human hepatocytes. Additionally, the study revealed that benzylpenicillin exhibited superior efficacy compared to ceftazidime and rifamycin SV as an antidote against α-amanitin (Magdalan et al., 2009[68]).

Patients on triple chemotherapy using MDAC, NAC, and penicillin G had the lowest mortality rate (8.3 %) among all assessed treatment plans (Trakulsrichai et al., 2017[111]).

Ceftazidine (Figure 9(Fig. 9)), a third-generation cephalosporin, has been significantly more effective than benzylpenicillin with fewer side effects. This antidote was the second most commonly used β-lactam, always in conjunction with silybin (Enjalbert et al., 2002[33]).

Silybum marianum (milk thistle) is the most extensively studied plant for treating liver disease. Its active compounds, such as silybin (Figure 10(Fig. 10)), silydianin, and silychristine, collectively called silymarin, are flavonolignans. Among them, silybin exhibits the highest biological activity, and milk thistle extracts are typically standardized to contain 70-80 % silybin (Luper, 1998[65]).

Silymarin's protective effects were examined in various experiments. Dogs received lethal doses of A. phalloides toxin. Administration of silybin (50 mg/kg) at 5 and 24 hours after poisoning mitigated serum changes (including Glutamyl oxaloacetic transaminase, Glutamyl pyruvic transaminase, alkaline phosphatase, and bilirubin) and prevented a decline in prothrombin time. The extent of hemorrhagic liver necrosis was significantly diminished (Vogel et al., 1984[113]).

The hepatoprotective action of silymarin was demonstrated by Desplaces et al., (1975[26]). Their research confirmed that an administration of 15 mg/kg of silymarin to animals (such as dogs, rabbits, rats, and mice) protected all animals when given 60 minutes before the toxin. Likewise, injecting silymarin at a dose of 100 mg/kg 10 minutes after A. phalloides exposure also resulted in complete protection (Desplaces et al., 1975[26]). However, in a murine model silybin was not effective in limiting hepatic injury after α-amanitin poisoning (Tong et al., 2007[105]). As previously described, Magdalan et al., (2010[67]) demonstrated that administering benzylpenicillin, N-acetylcysteine, and silybin to human hepatocyte cultures exhibited a similarly potent protective effect against amanitin toxicity. The cytoprotective effect of these antidotes wasn't dependent on the dose, indicating their high effectiveness (Magdalan et al., 2010[67]).

A total of 18 cases of Amanita palloides intoxication were treated by combined chemotherapy (Hruby et al., 1983[54]). All patients received silybin as standard treatment. It was noted that administering silybin even up to 48 hours after consuming the mushroom was an effective measure in preventing severe liver damage from amanita poisoning (Hruby et al., 1983[54]). Similarly, oral administration of silymarin was effective in 87.5 % of poisoning patients with hepatitis diagnosis (Trakulsrichai et al., 2017[111]).

The mechanism of action of silybin is still poorly understood. Silymarin and silybin hinder the absorption of toxins like phalloidin or α-amanitin, stopping them from attaching to cell surfaces and blocking membrane transport systems (Fraschini et al., 2002[37]).

Moreover, silymarin and silybin can alter the chemical and physical properties of cell membranes by interacting with their lipid components. Additionally, the proven ability of silymarin and silybin to neutralize free radicals can clarify their protective effects against liver-damaging substances. These antidotes likely work by countering free radicals and disrupting the lipid peroxidation processes implicated in liver injury caused by toxins. They may counteract the decline of two key antioxidants, glutathione (GSH) and superoxide dismutase (SOD), by reducing the burden of free radicals, boosting GSH levels, and enhancing SOD activity (Fraschini et al., 2002[37]).

Silymarin can penetrate the nucleus and influence RNA polymerase enzymes, leading to higher ribosomal production. This action holds significant therapeutic potential for mending harmed hepatocytes and reinstating normal liver functions (Pradhan and Girish, 2006[81]).

Thioctic acid (Figure 11(Fig. 11)), also known as α-lipoic acid, is an antioxidant compound. It collaborates with antioxidants such as vitamins C and E. Unlike other antioxidants, it possesses unique qualities that suggest it might be capable of substituting specific nutritional supplements. It promotes growth, aids energy production, and assists the liver in detoxifying the body. Thioctic acid also shields cells, regulates blood sugar and eliminates toxic metals from the bloodstream (Roy et al., 2021[93]).

Studies on thioctic acid's mechanism propose its potential for treating amatoxin hepatotoxicity. It acts as a free radical scavenger and could inhibit cell membrane lipid peroxidation by releasing hydrogen ions from dihydrothioctic acid, its primary metabolite (Enjalbert et al., 2002[33]).

The Instituto Seroterapico Italiano in Milan suggested that up to 300 mg of thioctic acid could be safely administered in a day through continuous infusion (Steyn, 1966[102]). Thirty-nine out of 40 patients were saved by this treatment in Ceske Budejovice in Bohemia (Steyn, 1966[102]). Thioctic acid infusions were used in the treatment of amanita mushroom poisoning in 75 patients between 1974 and 1978. While 10-50 % of patients recover without intervention, 89 percent (67 of 75) recover after lipoic acid infusion (Berkson, 1979[12]) A case involving a 32-year-old man who consumed A. phalloides highlighted thioctic acid's potential efficacy. Intravenous infusion of 300 mg within 24 hours, mixed with 10 percent glucose in water, led to the patient's improvement and symptom resolution on the following day (Becker et al., 1976[10]).

However, Poucheret et al., (2010[80]) reported that thioctic acid didn't significantly impact survival rates and showed limited therapeutic effects in the context of amatoxin poisoning. The lack of significant impact on patient survival by this compound may stem from its free radical scavenging effect not primarily targeting the main site of acute oxidative stress in amatoxin poisoning, which is the liver. Thioctic acid is known for potential side effects like hypoglycemia, which could worsen glucose imbalance induced by amatoxins. Additionally, it stimulates COX (cyclooxygenase) activity and related prostaglandin production, potentially conflicting with potent drugs like silybin if used together in treatments. These factors collectively contribute to thioctic acid's controversial role in amatoxin poisoning treatment (Poucheret et al., 2010[80]).

N-acetylcysteine (Figure 12(Fig. 12)) has been utilized in medicine for over 50 years as a mucolytic agent. It is recognized for addressing acetaminophen overdose and associated liver failure. Yet, N-acetylcysteine (NAC) serves as a versatile supplement, serving as a precursor to glutathione and elevating overall body glutathione levels (Tenório et al., 2021[103])

Acetaminophen transforms into harmful free radicals in the liver, binding to glutathione. NAC serves as a glutathione precursor and works as an effective therapy for acetaminophen overdose. Testing in mice indicated that NAC might not have a clinical role in treating A. phalloides ingestion, as its administration did not impact survival or liver enzyme elevation when compared to control animals (Schneider et al., 1992[97]). On the other hand, Magdalan et al., (2010[67]) demonstrated that exposing human hepatocyte cultures to NAC exhibited potent protection against α-amanitin-induced cytotoxic damage (Magdalan et al., 2010[67]).

According to Poucheret et al. (2010[80]), NAC operates on two levels. Firstly, it scavenges free radicals in a general manner. Secondly, its more targeted effect involves replenishing liver glutathione. Furthermore, NAC could potentially disrupt the intramolecular tryptathione bridge of amatoxin (critical for its toxicity). NAC administration is primarily associated with hepatoprotective function rather than an antidote role (Enjalbert et al., 2002[33]). A retrospective study found that the most frequently reported treatment (69.9 %) for amatoxin-intoxicated patients involved a combination of NAC and silybin (Lecot et al., 2023[62]). Consequently, further research is needed to thoroughly validate these mechanisms of action for NAC.

Cimetidine (Figure 13(Fig. 13)), a treatment typically used to reduce stomach acid production, has been studied for its ability to inhibit the uptake of amatoxins in the liver. By doing so, it may help mitigate the toxic effects of these compounds on the liver cells (Bang et al., 2022[7])

As a cytochrome P450 inhibitor, it was initially applied in amatoxin exposures due to the resemblance between amatoxin poisoning and other liver toxins that affect cytochrome P450 (Tong et al., 2007[105]).

Schneider et al. (1987[96]) studied cimetidine as an antidote against the key mushroom toxin α-amanitin. Examination of liver tissues from α-amanitin poisoned mice displayed significant mitochondrial alterations, but the hepatic mitochondria remained intact in mice treated with cimetidine either before exposure or within 6 hours (Schneider et al., 1987[96]).

However, it is important to note that while there is some experimental evidence suggesting cimetidine's potential benefits in amatoxin poisoning, further clinical research is needed to establish its effectiveness and safety in human cases.

In addition to established treatments for amatoxin poisoning, two potential promising antidotes have emerged: Polymyxin B (Pol) and cyclosporine A (CsA) (Figure 14(Fig. 14)). Experimental studies have shown that α-amanitin-intoxicated mice treated with Pol fully recover from toxicity, displaying reduced cellular degeneration and necrosis in the liver and kidney (Garcia et al., 2015[44]). When combined with methylprednisolone, Pol also lowered α-amanitin-induced liver damage, resulting in 100 % survival of treated animals (Garcia et al., 2019[41]). The suggested mechanism for Pol's effectiveness is its binding to RNAP II, thereby preventing α-amanitin from binding to the enzyme (Garcia et al., 2015[44]). However, in vitro studies did not observe any effects of Pol on α-amanitin cytotoxicity in cultured HepG2 cells (Rodrigues et al., 2020[91]) and there is currently no evidence for the efficacy of this potential antidote in humans intoxicated with α-amanitin.

On the other hand, CsA has also been explored for its potential to inhibit the OATP1B3 transporter, which prevents α-amanitin uptake by hepatocytes, thus averting hepatoxicity (Mackenzie et al., 2022[66]). Experimental animals treated with CsA showed hepatoxicity recovery, with 40 % survival when receiving CsA injections 4, 8, and 12 hours after α-amanitin exposure, and 100 % survival with simultaneous treatment. Additionally, CsA treatment reduced liver edema, necrosis, and cytoplasmic vacuolization (Garcia et al., 2022[39]). In human cases, a series of reports indicated that CsA administration (5 mg/kg IV over 4-6 hours every 24 hours) was effective in treating amatoxin ingestion in three patients. Moreover, the two patients with elevated aminotransferase levels fully regained normal liver enzyme values after CsA treatment (Mackenzie et al., 2022[66]).

Acute liver failure (ALF) is a severe syndrome where previously healthy individuals experience sudden and serious liver cell dysfunction. This can quickly lead to coma and death because of cerebral edema and failure of multiple organ systems (Larson 2008[61]). Nonetheless, not all A. phalloides poisoning patients experience acute liver failure, and not all cases result in fatality (Escudié et al., 2007[34]).

There are two surgical choices for liver transplantation: orthotopic liver transplantation and auxiliary liver transplantation. Orthotopic liver transplantation necessitates ongoing immunosuppression to sustain the graft. Auxiliary partial liver transplantation is an option when certain native livers can recover with partial hepatectomy and temporary support. The transplanted liver then offers temporary aid until the native liver regenerates (Barceloux, 2008[8]).

Timely and precise assessment of an individual patient is crucial in determining whether liver transplantation is necessary for treating fulminant hepatic failure. The Liver Unit at King's College Hospital has devised a prognostic model to identify patients with poor prognosis. The criteria for non-acetaminophen-induced fulminant hepatic failure include: prothrombin time > 100 seconds; any three of the following: age <10 or >40 years, jaundice >7 days before encephalopathy onset, prothrombin time >50 seconds, and bilirubin >300 μmol/L (O'Grady et al., 1989[77]). Furthermore, Escudié et al. (2007[34]) recommended that patients with an interval of <8 hours between ingestion and diarrhea should strongly consider liver transplantation. Encephalopathy need not be an absolute prerequisite for transplantation decision. Starting from day 4 of ingestion, a prothrombin index lower than 10 % alone is a reliable indicator for urgent transplantation (Escudié et al., 2007[34]).

Although definitive predictors are lacking, factors like encephalopathy stage, coagulation tests, metabolic irregularities, and age can aid in the strategic planning of liver transplantation (Enjalbert et al., 2002[33]).

This work is supported by National Funds by FCT-Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (https://doi.org/10.54499/UIDB/04033/2020) (accessed on 2 April 2024).

The authors declare that they have no conflict of interest.

Irene Gouvinhas thanks FCT (Fundação para a Ciência e Tecnologia) for funding through the Scientific Employment Stimulus-Individual Call (2022.00498.CEECIND), https://doi.org/10.54499/2022.00498.CEECIND/CP1749/CT0001 (accessed on 2 April 2024). Juliana Garcia and Jani Silva are grateful for funding support from Portuguese public funding through Investimento RE-C05-i02-Missão Interface N.º 01/C05-i02/2022 and from FCT for the projects titled “AquaValor-Centro de Valorização e Transferência de Tecnologia da Água” (NORTE-01-0246-FEDER-000053), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).