Human Poisoning from Poisonous Higher Fungi: Focus on Analytical Toxicology and Case Reports in Forensic Toxicology

Several families of higher fungi contain mycotoxins that cause serious or even fatal poisoning when consumed by humans. The aim of this review is to inventory, from an analytical point of view, poisoning cases linked with certain significantly toxic mycotoxins: orellanine, α- and β-amanitin, muscarine, ibotenic acid and muscimol, and gyromitrin. Clinicians are calling for the cases to be documented by toxicological analysis. This document is therefore a review of poisoning cases involving these mycotoxins reported in the literature and carries out an inventory of the analytical techniques available for their identification and quantification. It seems indeed that these poisonings are only rarely documented by toxicological analysis, due mainly to a lack of analytical methods in biological matrices. There are many reasons for this issue: the numerous varieties of mushroom involved, mycotoxins with different chemical structures, a lack of knowledge about distribution and metabolism. To sum up, we are faced with (i) obstacles to the documentation and interpretation of fatal (or non-fatal) poisoning cases and (ii) a real need for analytical methods of identifying and quantifying these mycotoxins (and their metabolites) in biological matrices.


Introduction
There is an extremely diverse range of fungi about which little is known. One million five hundred thousand species were known in 2002, 5.1 million in 2005, and the figure reached 13.5 million species in 2018. In reality, the exact number of fungal species on Earth is as yet unknown, since we are only aware of a tiny proportion of this diversity, of which only 100,000 species have been described [1]. Among these, there are about 5000 species of so-called higher fungi [2], those where the sporophore (the reproductive organ in fungi) is visible to the naked eye. Of these, a few dozen species of mushroom [1] contain mycotoxins, which, when ingested, could cause poisoning of varying degrees of severity and may even result in death. These poisonings can be classified according to 14 specific syndromes, some more serious than others: acromelalgic, cerebellar, coprinic, digestive (and resinoid), encephalopathy, gyromitrin, muscarinic, orellanus, pantherina, paxillus, phalloidin, proximien, psilocybin (or narcotic), and rhabdomyolysis syndrome [3,4].

Toxic Mechanism and Toxicity in Humans and/or Animals
The toxicity of orellanine lies in its strong nephrotic properties leading to acute renal failure (group 1C in the White et al. classification [5]). Its toxic mechanism has not been precisely established yet. However, Richard and his team have shown that orellanine is responsible for the inhibition of proteins in the cytoplasm and mitochondria of renal cells after tests on Madin-Darby canine renal cells [17]. Other hypotheses have been advanced such as the inhibition of DNA and RNA in the renal cells, glutathione depletion, or inhibition of mitochondrial adenosine triphosphate production [16,18].
There is high variability in clinical outcomes in the case of poisoning: the evolution can be spontaneously favorable or can deteriorate into chronic renal failure, requiring a kidney transplant [19]. There is no antidote for orellanine; treatment is symptomatic (hemodialysis, N-acetylcysteine, and steroids) [7,19,20]. Several studies in mice show that the oral median lethal dose (LD50) is between 30 and 90 mg/kg [21,22]. However, humans have been shown to be far more sensitive than mice to this mycotoxin. In practice, the ingestion of 6 mushrooms can lead to acute renal failure requiring dialysis [23].

Toxic Species
Orellanine is the main toxin found in mushrooms of the genus Cortinarius of the family Cortinariaceae. The most frequently reported in poisoning cases are C. orellanus [24,25] (Figure 2) and C. speciosissimus [7,19]. Some cases also mention C. orellanosus [23], C. armillatus [26], and C. eartoxicus [27]. The toxicity of C. splendens [28] is still in doubt. These species are mainly found in Europe and North America. Some cases of poisoning in Australia have also been reported [27,29].

Toxic Species
Orellanine is the main toxin found in mushrooms of the genus Cortinarius of the family Cortinariaceae. The most frequently reported in poisoning cases are C. orellanus [24,25] (Figure 2) and C. speciosissimus [7,19]. Some cases also mention C. orellanosus [23], C. armillatus [26], and C. eartoxicus [27]. The toxicity of C. splendens [28] is still in doubt. These species are mainly found in Europe and North America. Some cases of poisoning in Australia have also been reported [27,29].

Toxic Mechanism and Toxicity in Humans and/or Animals
The toxicity of orellanine lies in its strong nephrotic properties leading to acute renal failure (group 1C in the White et al. classification [5]). Its toxic mechanism has not been precisely established yet. However, Richard and his team have shown that orellanine is responsible for the inhibition of proteins in the cytoplasm and mitochondria of renal cells after tests on Madin-Darby canine renal cells [17]. Other hypotheses have been advanced such as the inhibition of DNA and RNA in the renal cells, glutathione depletion, or inhibition of mitochondrial adenosine triphosphate production [16,18].
There is high variability in clinical outcomes in the case of poisoning: the evolution can be spontaneously favorable or can deteriorate into chronic renal failure, requiring a kidney transplant [19]. There is no antidote for orellanine; treatment is symptomatic (hemodialysis, N-acetylcysteine, and steroids) [7,19,20]. Several studies in mice show that the oral median lethal dose (LD50) is between 30 and 90 mg/kg [21,22]. However, humans have been shown to be far more sensitive than mice to this mycotoxin. In practice, the ingestion of 6 mushrooms can lead to acute renal failure requiring dialysis [23].

Toxic Species
Orellanine is the main toxin found in mushrooms of the genus Cortinarius of the family Cortinariaceae. The most frequently reported in poisoning cases are C. orellanus [24,25] (Figure 2) and C. speciosissimus [7,19]. Some cases also mention C. orellanosus [23], C. armillatus [26], and C. eartoxicus [27]. The toxicity of C. splendens [28] is still in doubt. These species are mainly found in Europe and North America. Some cases of poisoning in Australia have also been reported [27,29].

Description of the Syndrome
Orellanine causes orellanus syndrome, which is characterized by a long latency period: between 2-4 and 14 days after ingestion [16]. To date, there is no scientific explanation for this exceptionally long latency period. The fact remains that this sometimes makes it difficult to link the ingestion with the clinical phase of poisoning. The first symptoms to appear are usually nausea, vomiting, diarrhea, stomach pains, extreme thirst, headaches, anuria, or polyuria depending on the case (cf. Table 5). These symptoms are followed by renal impairment necessitating transplantation. If left untreated, the patient may die of acute renal failure.

Human Poisoning Cases Reported
Many cases of orellanine poisoning have been reported in the literature since 1957. A number of them are listed nonexhaustively in Table 5. These cases include 27 reported deaths and 17 kidney transplants in people aged 14 and 60. Most poisonings are unintentional, sometimes by confusion with hallucinogenic mushrooms [29,31]. One case reports voluntary consumption of Cortinarius orellanus by a psychiatric patient [24]. Due to its long latency period, many patients consume mushrooms several times, sometimes a few days after the first meal [7,32,33]. The majority of patients have a serum creatinine over the physiological range at the arrival to the hospital. Those with a higher level underwent a renal transplantation.

Analytical Aspect
Research began in the late 1970s to develop a quick, sensitive, and reliable analytical method for identifying and quantifying orellanine in mushrooms as a first step, then in biological matrices such as blood, urine, or organs (cf. Table 2). Many methods are based on the thin layer chromatography, only one is based on the gas chromatography. Most recent methods consist of a liquid chromatography coupled with tandem mass spectrometry.       Many poisoning cases in the biological matrices documented by research for orellanine have revealed the absence of orellanine in urine, plasma, and dialysis fluids between 2 and 25 days after the ingestion of mushrooms [41]. However, Rapior et al. using thin layer chromatography coupled with spectrofluorometry, reported a concentration of 6.12 mg/L in plasma 10 days after the ingestion of mushrooms [24]. Orellanine has also been quantified several times in renal biopsies with concentrations between 35 and 3000 mg/L up to 180 days after poisoning [24,41].

Toxic Compounds
Since the 1790s (Paulet's research into the toxins of Amanita phalloides, 1793-1808) [57], researchers have taken an interest in the compounds responsible for the toxicity of A. phalloides. After the identification of other compounds contained in these mushrooms (e.g., phalloidin), Wieland et al. first isolated an amanitin in 1941 (which they later named α-amanitin) then 8 other amatoxins were isolated and their structure described (β-amanitin, γ-amanitin, ε-amanitin, amanin, amanullin, amaninamide, amanullinic acid, and proamanullin) [57]. The main toxins of certain mushrooms in this family are α-amanitin and β-amanitin. α-amanitin (C 39  Many poisoning cases in the biological matrices documented by research for orellanine have revealed the absence of orellanine in urine, plasma, and dialysis fluids between 2 and 25 days after the ingestion of mushrooms [41]. However, Rapior et al. using thin layer chromatography coupled with spectrofluorometry, reported a concentration of 6.12 mg/L in plasma 10 days after the ingestion of mushrooms [24]. Orellanine has also been quantified several times in renal biopsies with concentrations between 35 and 3000 mg/L up to 180 days after poisoning [24,41].

Toxic Mechanism and Toxicity in Humans and/or Animals
In the new classification, the amatoxins are classified in the cytotoxic group (1A) [5] as they are responsible for inhibiting RNA polymerase II and the transcription of DNA into RNA by interfering with messenger RNA. This brings about inhibition of protein synthesis, which leads to cell necrosis. The first cells to be affected are those with a high rate of protein synthesis such as enterocytes, hepatocytes and proximal renal cells [59]. Studies in mice show that renal lesions only occur in poisoning with low levels of amatoxins. In poisoning cases with high levels, the subject die due to acute liver failure or hypoglycemia before the renal lesions appear [60,61]. Amatoxins are mainly eliminated in the bile, but there is an enterohepatic cycle, which prolongs the hepatoxic action [62]. The amatoxins are not thermosensitive, which means they cannot be destroyed by either cooking or freezing the mushrooms [58]. Moreover, they are gastroresistant [58] and their metabolism is currently unknown.

Toxic Mechanism and Toxicity in Humans and/or Animals
In the new classification, the amatoxins are classified in the cytotoxic group (1A) [5] as they are responsible for inhibiting RNA polymerase II and the transcription of DNA into RNA by interfering with messenger RNA. This brings about inhibition of protein synthesis, which leads to cell necrosis. The first cells to be affected are those with a high rate of protein synthesis such as enterocytes, hepatocytes and proximal renal cells [59]. Studies in mice show that renal lesions only occur in poisoning with low levels of amatoxins. In poisoning cases with high levels, the subject die due to acute liver failure or hypoglycemia before the renal lesions appear [60,61]. Amatoxins are mainly eliminated in the bile, but there is an enterohepatic cycle, which prolongs the hepatoxic action [62].
Several studies show that the LD 50 of α-amanitin in humans is estimated to be 0.1 mg/kg per os [63]. Bearing in mind that a sporophore of Amanita phalloides (20-25 g) can contain 5-8 mg of amatoxins [64], the ingestion of one A. phalloides mushroom is theoretically a lethal dose for a 75 kg man. The same order of magnitude is found in mice in a study published by Wieland in 1959 [57] (LD 50 = 0.1 mg/kg for α-amanitin and 0.4 mg/kg for β-amanitin by intraperitoneal injection). Finally, it has been shown that the concentration of amatoxins in the mushroom increases during the first stages of the mushroom's development, then decreases during the mature stage [65].
As with orellanine, no specific antidote exists for the amanitins. Treatment is symptomatic (dialysis, activated charcoal hemoperfusion, glucose/saline perfusion, etc.) [66,67]. Only kidney or liver transplantation (depending on the symptoms) can save a patient with multiple organ failure [67,68]. Some authors propose treatments such as thioctic acid (alpha lipoic acid) [69,70], penicillin G [71], or silibinin [72,73], which may be capable of limiting, if not inhibiting, the amatoxins' penetration into the liver cells and/or interrupting the enterohepatic cycle of the toxins [74]. However, these treatments have not really been clinically proven and there is no evidence to support the use of penicillin G or of thioctic acid. They are therefore not considered as part of the protocol for treatment of amanitin poisoning.
In view of all the cases of amanitin poisoning reported in the literature, it seems clear that infants and small children are more sensitive to these mycotoxins than adults, probably because of their lower body mass: the same dose of toxins ingested will be more toxic and the percentage of fatalities will be higher in young subjects.

Toxic Species
The amatoxins are the compounds responsible for the toxicity of Amanita phalloides [57] (Figure 4) also known as "death cap" in English-speaking countries [58], and without doubt the most well-known poisonous mushroom in the world. Probably all members of section Phalloideae contain potentially lethal levels of amanitins. These mycotoxins are also found in other species such as A. verna [75] and A. virosa [62], A. bisporigera [76], and A. ocreata [77]. Other genera contain amatoxins including Galerina (G. marginata and G. autumnalis) and Lepiota (L. brunneoincarnata and L. helveola) within the main species of concern [78].
Amatoxin-containing mushroom species have been worldwide identified (Northern, Central, and Western Europe, North and South America, South-East Asia, and Northern and Southern Africa) [58].
It should be noted that Amanita phalloides contains two other groups of toxins: phallotoxins and virotoxins [58]. These two families of cyclic peptides are only toxic by parenteral administration as they are hardly (or not at all) absorbed by the gastrointestinal tract [58]. They are therefore not usually taken into consideration in Amanita phalloides poisoning.

Description of the Syndrome
The amatoxins are responsible for phalloidin syndrome, which, like orellanus syndrome, is characterized by a long latency period (between 6 and 24 h) after ingestion of the mushroom [58]. First occurring symptoms are gastrointestinal (nausea, vomiting, diarrhea, and stomach pains) over a period of about 24 h. The second stage is a period of remission, usually lasting 24-36 h. During this period, the serum activity levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) rise progressively, showing liver damage. The third stage is characterized by renal and hepatic impairment, which could result in hepatic encephalopathy, convulsions, coma and death (4-7 days after ingestion of mushrooms) [74]. Death by amatoxin poisoning is most often caused by liver, or kidney failure, or sometimes both (cf. Table 3). 4) also known as "death cap" in English-speaking countries [58], and without doubt the most wellknown poisonous mushroom in the world. Probably all members of section Phalloideae contain potentially lethal levels of amanitins. These mycotoxins are also found in other species such as A. verna [75] and A. virosa [62], A. bisporigera [76], and A. ocreata [77]. Other genera contain amatoxins including Galerina (G. marginata and G. autumnalis) and Lepiota (L. brunneoincarnata and L. helveola) within the main species of concern [78].

Human Poisoning Cases Reported
Given the large number of mushroom species containing amanitins throughout the world, a great number of amatoxin poisoning cases have been reported in the literature since the beginning of the last century (Table 3). Of these recorded poisonings, 72 deaths and 33 liver transplants are listed. Five of the deaths occurred up to several months after liver transplantation. This suggests persistent toxicity capable of damaging the graft. One case is unusual, the patient ate 2 caps of Amanita phalloides only in order to test the toxicity [114].
The result is fatal in 10-30% of cases [58], with the percentage tending to decrease mainly due to liver transplantation.

Analytical Aspect
Research began in the mid-1970s to develop a sensitive and reliable analytical method for identifying and quantifying αand β-amanitin through radioimmunological techniques, thin layer chromatography or liquid chromatography-UV detection. Technological developments over the years have enabled researchers to reach better and better sensitivity levels using high-resolution mass detectors (cf. Table 7).       Testing for amanitins in various biological samples in a known case of amatoxin poisoning has revealed the elimination kinetics of these compounds. It is possible to find amanitins in blood (plasma or serum) up to 36-48 h after ingestion [61,90,151] in concentrations varying from 10 to 200 µg/L [91] and in urine up to 96 h after ingestion [89,151]. The urine concentrations range from 1 to 7100 µg/L, with a peak between 24 and 72 h [90,140,151]. Jaeger et al. have shown that it is also possible to find high concentrations of αand β-amanitin in gastroduodenal fluid and feces (between 208 and 4950 µg/L in gastroduodenal fluid and between 23 and 14,900 µg/L in feces) [90].
The amanitins have hepatic and renal tropism. As a consequence, it should be of interest to assay them in these matrices. Jaeger et al. reported concentrations of 10-3298 µg/L found in the liver and kidney samples (from autopsy or biopsy) of poisoned subjects [90].
There is an immunological technique for assaying alpha and gamma amanitins (but not beta amanitin) in urine available as a kit (BÜHLMANN ELISA kit). Its limit of detection is 0.22 µg/L with a limit of quantification of 1.5 µg/L [152].

Toxic Compounds
The first attempt to isolate muscarine, which was considered the main active substance in Amanita muscaria [153], dates back to the early 1810s with Braconnot and Schrader. At that time several researchers had tried in vain to isolate this psychoactive compound. It was not until 1869 that Schmiedeberg and Koppe believed they had isolated the substance and called it muscarine. The substance they isolated proved to be a mixture of muscarine and choline. Pure muscarine was actually isolated for the first time by King in 1922 [154]. The structure of muscarine was proposed in 1957 by Kögl et al. [155]: C 9 H 20 NO 2 + , M = 174.3 ( Figure 5).
Muscarine (tetrahydro-4-hydroxy-N,N,N-5-tetramethyl-2-furanmethanaminium) is a water-soluble thermostable alkaloid [154]. To the best of our knowledge, no studies or metabolism data have been published about this mycotoxin. Testing for amanitins in various biological samples in a known case of amatoxin poisoning has revealed the elimination kinetics of these compounds. It is possible to find amanitins in blood (plasma or serum) up to 36-48 h after ingestion [61,90,151] in concentrations varying from 10 to 200 µg/L [91] and in urine up to 96 h after ingestion [89,151]. The urine concentrations range from 1 to 7100 µg/L, with a peak between 24 and 72 h [90,140,151]. Jaeger et al. have shown that it is also possible to find high concentrations of α-and β-amanitin in gastroduodenal fluid and feces (between 208 and 4950 µg/L in gastroduodenal fluid and between 23 and 14900 µg/L in feces) [90].
The amanitins have hepatic and renal tropism. As a consequence, it should be of interest to assay them in these matrices. Jaeger et al. reported concentrations of 10-3298 µg/L found in the liver and kidney samples (from autopsy or biopsy) of poisoned subjects [90].
There is an immunological technique for assaying alpha and gamma amanitins (but not beta amanitin) in urine available as a kit (BÜHLMANN ELISA kit). Its limit of detection is 0.22 µg/L with a limit of quantification of 1.5 µg/L [152].

Toxic Compounds
The first attempt to isolate muscarine, which was considered the main active substance in Amanita muscaria [153], dates back to the early 1810s with Braconnot and Schrader. At that time several researchers had tried in vain to isolate this psychoactive compound. It was not until 1869 that Schmiedeberg and Koppe believed they had isolated the substance and called it muscarine. The substance they isolated proved to be a mixture of muscarine and choline. Pure muscarine was actually isolated for the first time by King in 1922 [154]. The structure of muscarine was proposed in 1957 by Kögl et al. [155]: C9H20NO2 + , M = 174.3 ( Figure 5). Muscarine (tetrahydro-4-hydroxy-N,N,N-5-tetramethyl-2-furanmethanaminium) is a water-soluble thermostable alkaloid [154]. To the best of our knowledge, no studies or metabolism data have been published about this mycotoxin.

Toxic Mechanism and Toxicity in Humans and/or Animals
Muscarine is an agonist for the neurotransmitter acetylcholine; it activates muscarinic acetylcholine receptors and thereby activates the parasympathetic nervous system [155]. Due to its positively charged quaternary amine group, muscarine does not cross the blood-brain barrier and therefore does not reach the central nervous system. This mechanism of action puts it in group 2B of the White et al. classification [5] (neurotoxic molecules that do not reach the central nervous system). Unlike many mycotoxins, there is an antidote to muscarine: atropine. Administered intravenously, atropine counters the toxic cardiac effects of muscarine [156]. Muscarine poisoning must be proven (for example by identifying the mushroom species ingested) before administering atropine, since atropine can exacerbate some symptoms if administered in error (see ibotenic acid and muscimol, below).
The toxic effects of muscarine vary according to the amount ingested. Muscarine poisoning is rarely fatal; patients with pre-existing cardiac disorders will be more sensitive. The symptomatology

Toxic Mechanism and Toxicity in Humans and/or Animals
Muscarine is an agonist for the neurotransmitter acetylcholine; it activates muscarinic acetylcholine receptors and thereby activates the parasympathetic nervous system [155]. Due to its positively charged quaternary amine group, muscarine does not cross the blood-brain barrier and therefore does not reach the central nervous system. This mechanism of action puts it in group 2B of the White et al. classification [5] (neurotoxic molecules that do not reach the central nervous system). Unlike many mycotoxins, there is an antidote to muscarine: atropine. Administered intravenously, atropine counters the toxic cardiac effects of muscarine [156]. Muscarine poisoning must be proven (for example by identifying the mushroom species ingested) before administering atropine, since atropine can exacerbate some symptoms if administered in error (see ibotenic acid and muscimol, below).
The toxic effects of muscarine vary according to the amount ingested. Muscarine poisoning is rarely fatal; patients with pre-existing cardiac disorders will be more sensitive. The symptomatology usually resolves after a few hours. In cases where the patient is severely dehydrated, compensation for fluid and electrolyte loss should be considered [2].
Toxicity studies show the i.v. LD 50 of muscarine in mice is 0.23 mg/kg [157,158]. No numerical data for humans have been published.
No mechanism or preferential route of elimination of muscarine from the organism has been described in the literature.

Toxic Species
Muscarine is actively present in several mushroom families: around 40 Inocybes of the family Inocybaceae (I. erubescens, I. subdestricta, I. fastigiata, I. geophilla, etc.), around 15 Clytocybes (Figure 6) of the family Tricholomataceae (C. cerussata, C. dealbata, C. rivulosa, C. phylophilla, etc.) [156]. It is also found in the genus Amanita (A. muscaria and A. pantherina) but in minute quantities [159], which makes its toxic action insignificant compared with these mushrooms' other active compounds. Amanita muscaria takes its name from muscarine since, as explained above, muscarine was isolated from this species. However, the fly agaric only contains 0.0002-0.0003% of muscarine [153,159,160]. By comparison, I. subdestricta contains 0.43% and C. dealbata 0.15% [153]. usually resolves after a few hours. In cases where the patient is severely dehydrated, compensation for fluid and electrolyte loss should be considered [2]. Toxicity studies show the i.v. LD50 of muscarine in mice is 0.23 mg/kg [157,158]. No numerical data for humans have been published.
No mechanism or preferential route of elimination of muscarine from the organism has been described in the literature.

Toxic Species
Muscarine is actively present in several mushroom families: around 40 Inocybes of the family Inocybaceae (I. erubescens, I. subdestricta, I. fastigiata, I. geophilla, etc.), around 15 Clytocybes (Figure 6) of the family Tricholomataceae (C. cerussata, C. dealbata, C. rivulosa, C. phylophilla, etc.) [156]. It is also found in the genus Amanita (A. muscaria and A. pantherina) but in minute quantities [159], which makes its toxic action insignificant compared with these mushrooms' other active compounds. Amanita muscaria takes its name from muscarine since, as explained above, muscarine was isolated from this species. However, the fly agaric only contains 0.0002-0.0003% of muscarine [153,159,160]. By comparison, I. subdestricta contains 0.43% and C. dealbata 0.15% [153]. Due to the great diversity of mushrooms containing muscarine, the toxin has been identified on every continent.

Description of the Syndrome
The syndrome associated with muscarine is called muscarinic syndrome. It has a short latency period (<6 h) as the first symptoms appear between 15 min and 2 h after ingestion [156]. The main clinical signs of muscarine poisoning are gastrointestinal distress (nausea, vomiting, diarrhea, and stomach pains), extreme sweating, bronchial, salivary and ocular hypersecretion, and blurred vision. Observed bradycardia, hypotension, and miosis are the direct consequences of acetylcholine receptors activation. In the most severe cases muscarine can cause myoclonus, convulsions, and loss of consciousness that may lead to coma and the death of the patient (cf. Table 5). Due to the great diversity of mushrooms containing muscarine, the toxin has been identified on every continent.

Description of the Syndrome
The syndrome associated with muscarine is called muscarinic syndrome. It has a short latency period (<6 h) as the first symptoms appear between 15 min and 2 h after ingestion [156]. The main clinical signs of muscarine poisoning are gastrointestinal distress (nausea, vomiting, diarrhea, and stomach pains), extreme sweating, bronchial, salivary and ocular hypersecretion, and blurred vision. Observed bradycardia, hypotension, and miosis are the direct consequences of acetylcholine receptors activation. In the most severe cases muscarine can cause myoclonus, convulsions, and loss of consciousness that may lead to coma and the death of the patient (cf. Table 5).

Human Poisoning Cases Reported
Case reports about muscarine poisoning are relatively rare. Table 5 shows published cases of muscarine poisoning. A fatal outcome was observed in three cases: an 11-year-old child [174], a 67-year-old woman presenting comorbidities (diabetes, arterial hypertension, and respiratory insufficiency) [172], and a 53-year-old woman with no particular medical history [170]. The other cases present a positive outcome.

Analytical Aspect
Since muscarine was isolated in 1922 [154], few analytical techniques have been published for identifying and quantifying the compound in different matrices. The first published techniques used thin layer chromatography or gas chromatography with mass detection for qualitative and/or quantitative analysis of muscarine in mushrooms. The technological advances of the early 21st century have enabled considerably greater sensitivity with liquid chromatography techniques coupled to tandem mass spectrometry. With these techniques it is now possible to quantify muscarine in biological matrices such as urine (Table 6).
To the best of our knowledge, no research on muscarine in blood or any other biological matrix has been published. Only one publication mentions a numerical value for muscarine in urine: 0.045 mg/L of muscarine was found in the urine of a 55-year-old suspected of having ingested A. muscaria [155].

Toxic Compounds
Ibotenic acid or α-amino-3-hydroxy-5-isoxazoleacetic acid (C 5 H 6 N 2 O 4 , M = 158.1) is an alkaloid, which is degraded by decarboxylation into muscimol (3-hydroxy-5-aminomethylisoxazole, C 4 H 6 N 2 O 2 , M = 114.1; Figures 7 and 8). These compounds, isolated and described in the 1960s by a Japanese team, are thermostable [153] but the dehydration of ibotenic acid leads to the formation of muscimol by decarboxylation [183]. It would therefore be logical to consider the toxicity of cooked A. muscaria and A. pantherina mushrooms to be mainly attributable to muscimol. These two mycotoxins are the major factors in poisoning, but other toxins have also been identified in the mushrooms, including muscarine, in very low quantities, and muscazone, a structural isomer of ibotenic acid with less potent psychoactive properties than muscimol or ibotenic acid [153,183].

Toxic Compounds
Ibotenic acid or α-amino-3-hydroxy-5-isoxazoleacetic acid (C5H6N2O4, M = 158.1) is an alkaloid, which is degraded by decarboxylation into muscimol (3-hydroxy-5-aminomethylisoxazole, C4H6N2O2, M = 114.1; Figures 7 and 8). These compounds, isolated and described in the 1960s by a Japanese team, are thermostable [153] but the dehydration of ibotenic acid leads to the formation of muscimol by decarboxylation [183]. It would therefore be logical to consider the toxicity of cooked A. muscaria and A. pantherina mushrooms to be mainly attributable to muscimol. These two mycotoxins are the major factors in poisoning, but other toxins have also been identified in the mushrooms, including muscarine, in very low quantities, and muscazone, a structural isomer of ibotenic acid with less potent psychoactive properties than muscimol or ibotenic acid [153,183].  DeFeudis [160] states that muscimol is metabolized quickly after ingestion, and that consequently, its toxicity is shared with its psychoactive metabolites. However, no concrete metabolic study has been published about muscimol or ibotenic acid.

Toxic Mechanism and Toxicity in Humans and/or Animals
Ibotenic acid and muscimol are isoxazoles derived from glutamic acid and γ-aminobutyric acid (GABA) respectively [183]. Ibotenic acid and muscimol can cross the blood-brain barrier and thus act on the central nervous system [184], which puts them in group 2C of the White et al. classification [5] (neurotoxic molecules that reach the central nervous system). Ibotenic acid is a glutamate neurotransmitter agonist, a powerful neuronal excitant. It acts on the glutamic acid receptors associated with memory and learning. Muscimol is a γ-aminobutyric acid (GABA) agonist. It acts on the GABA receptors with a depressant effect and therefore causes related toxic effects such as visual distortions/hallucinations, loss of balance, slight muscle contractions, and altered sensory perceptions [153,183]. These two alkaloids are preferentially eliminated in urine [153,183]. Ibotenic acid and muscimol can be detected in urine one hour after mushroom ingestion [153].
Fatal poisoning by ibotenic acid and muscimol is very rare [153]. There is no antidote; the only treatment is symptomatic. Hospitalization for neurological surveillance is recommended [156]. In

Toxic Compounds
Ibotenic acid or α-amino-3-hydroxy-5-isoxazoleacetic acid (C5H6N2O4, M = 158.1) is an alkaloid, which is degraded by decarboxylation into muscimol (3-hydroxy-5-aminomethylisoxazole, C4H6N2O2, M = 114.1; Figures 7 and 8). These compounds, isolated and described in the 1960s by a Japanese team, are thermostable [153] but the dehydration of ibotenic acid leads to the formation of muscimol by decarboxylation [183]. It would therefore be logical to consider the toxicity of cooked A. muscaria and A. pantherina mushrooms to be mainly attributable to muscimol. These two mycotoxins are the major factors in poisoning, but other toxins have also been identified in the mushrooms, including muscarine, in very low quantities, and muscazone, a structural isomer of ibotenic acid with less potent psychoactive properties than muscimol or ibotenic acid [153,183].  DeFeudis [160] states that muscimol is metabolized quickly after ingestion, and that consequently, its toxicity is shared with its psychoactive metabolites. However, no concrete metabolic study has been published about muscimol or ibotenic acid.

Toxic Mechanism and Toxicity in Humans and/or Animals
Ibotenic acid and muscimol are isoxazoles derived from glutamic acid and γ-aminobutyric acid (GABA) respectively [183]. Ibotenic acid and muscimol can cross the blood-brain barrier and thus act on the central nervous system [184], which puts them in group 2C of the White et al. classification [5] (neurotoxic molecules that reach the central nervous system). Ibotenic acid is a glutamate neurotransmitter agonist, a powerful neuronal excitant. It acts on the glutamic acid receptors associated with memory and learning. Muscimol is a γ-aminobutyric acid (GABA) agonist. It acts on the GABA receptors with a depressant effect and therefore causes related toxic effects such as visual distortions/hallucinations, loss of balance, slight muscle contractions, and altered sensory perceptions [153,183]. These two alkaloids are preferentially eliminated in urine [153,183]. Ibotenic acid and muscimol can be detected in urine one hour after mushroom ingestion [153].
Fatal poisoning by ibotenic acid and muscimol is very rare [153]. There is no antidote; the only treatment is symptomatic. Hospitalization for neurological surveillance is recommended [156]. In DeFeudis [160] states that muscimol is metabolized quickly after ingestion, and that consequently, its toxicity is shared with its psychoactive metabolites. However, no concrete metabolic study has been published about muscimol or ibotenic acid.

Toxic Mechanism and Toxicity in Humans and/or Animals
Ibotenic acid and muscimol are isoxazoles derived from glutamic acid and γ-aminobutyric acid (GABA) respectively [183]. Ibotenic acid and muscimol can cross the blood-brain barrier and thus act on the central nervous system [184], which puts them in group 2C of the White et al. classification [5] (neurotoxic molecules that reach the central nervous system). Ibotenic acid is a glutamate neurotransmitter agonist, a powerful neuronal excitant. It acts on the glutamic acid receptors associated with memory and learning. Muscimol is a γ-aminobutyric acid (GABA) agonist. It acts on the GABA receptors with a depressant effect and therefore causes related toxic effects such as visual distortions/hallucinations, loss of balance, slight muscle contractions, and altered sensory perceptions [153,183]. These two alkaloids are preferentially eliminated in urine [153,183]. Ibotenic acid and muscimol can be detected in urine one hour after mushroom ingestion [153].
Fatal poisoning by ibotenic acid and muscimol is very rare [153]. There is no antidote; the only treatment is symptomatic. Hospitalization for neurological surveillance is recommended [156]. In some cases it is necessary to sedate the patient to manage excessive agitation [9,162]. Atropine is to be avoided as it has a similar action to ibotenic acid and muscimol.
Ibotenic acid and muscimol are lethal in very high doses. The LD 50 in rats is 129 mg/kg for ibotenic acid and 45 mg/kg for muscimol [158,185,186]. Stebelska [185] refers to a study of the toxicity of isoxazoles on mammals: the oral LD 50 for muscimol is 10 mg/kg in rabbits and the oral LD 50 for ibotenic acid is 38 mg/kg in mice. As with muscarine, no data for humans have yet been published. A sporophore of Amanita muscaria can contain between 292 and 6570 µg/g of ibotenic acid and between 73 and 2440 µg/g of muscimol [187]. Given the average weight of 60 g and the minimal dose to produce psychotropic effects of 30-60 mg of ibotenic acid and around 6-10 mg of muscimol, a single mushroom is enough to experience hallucinogenic effects [185]. Some studies have shown that the intensity of the effects varies according to which part of the mushroom is consumed. Indeed, the cap of the mushroom has a higher concentration of psychoactive substances than the stem [188,189].

Toxic Species
Ibotenic acid and muscimol are mainly found in Amanita muscaria ( Figure 9) and Amanita pantherina mushrooms, which belong to the genus Amanita of the family Amanitaceae. Virtually all mushrooms in genus Amanita contain high levels of muscimol and ibotenic acid. A. muscaria is undoubtedly the most iconic mushroom in the world, represented in illustrations, cartoons, etc., due to its bright colors and white spotted cap. These mushrooms have been identified in the United States, sub-Saharan Africa (South Africa, Zimbabwe) Japan, and Europe (cf. Table 5).
Pharmaceuticals 2020, 13, x FOR PEER REVIEW 47 of 66 some cases it is necessary to sedate the patient to manage excessive agitation [9,162]. Atropine is to be avoided as it has a similar action to ibotenic acid and muscimol. Ibotenic acid and muscimol are lethal in very high doses. The LD50 in rats is 129 mg/kg for ibotenic acid and 45 mg/kg for muscimol [158,185,186]. Stebelska [185] refers to a study of the toxicity of isoxazoles on mammals: the oral LD50 for muscimol is 10 mg/kg in rabbits and the oral LD50 for ibotenic acid is 38 mg/kg in mice. As with muscarine, no data for humans have yet been published.
A sporophore of Amanita muscaria can contain between 292 and 6570 µg/g of ibotenic acid and between 73 and 2440 µg/g of muscimol [187]. Given the average weight of 60 g and the minimal dose to produce psychotropic effects of 30-60 mg of ibotenic acid and around 6-10 mg of muscimol, a single mushroom is enough to experience hallucinogenic effects [185]. Some studies have shown that the intensity of the effects varies according to which part of the mushroom is consumed. Indeed, the cap of the mushroom has a higher concentration of psychoactive substances than the stem [188,189].

Toxic Species
Ibotenic acid and muscimol are mainly found in Amanita muscaria ( Figure 9) and Amanita pantherina mushrooms, which belong to the genus Amanita of the family Amanitaceae. Virtually all mushrooms in genus Amanita contain high levels of muscimol and ibotenic acid. A. muscaria is undoubtedly the most iconic mushroom in the world, represented in illustrations, cartoons, etc., due to its bright colors and white spotted cap. These mushrooms have been identified in the United States, sub-Saharan Africa (South Africa, Zimbabwe) Japan, and Europe (cf. Table 5). Figure 9. Amanita muscaria [190].
The possession, purchase, and sale of ibotenic acid and muscimol are not regulated in France. However, the possession, purchase, and sale of Amanita muscaria are illegal in the Netherlands [191], the state of Louisiana in the USA, the UK [192], and Romania [192]. In Thailand hallucinogenic mushrooms are classified as class V narcotics and are therefore illegal [193]. In Japan these two mushroom species are sold openly as dried mushrooms or dried mushroom "powder" on the internet and in "smoke shops" [186].

Description of the Syndrome
The syndrome produced by consuming mushrooms containing ibotenic acid and muscimol is called pantherina syndrome (or myco-atropine syndrome) [156]. The syndrome is characterized by a Figure 9. Amanita muscaria [190].
The possession, purchase, and sale of ibotenic acid and muscimol are not regulated in France. However, the possession, purchase, and sale of Amanita muscaria are illegal in the Netherlands [191], the state of Louisiana in the USA, the UK [192], and Romania [192]. In Thailand hallucinogenic mushrooms are classified as class V narcotics and are therefore illegal [193]. In Japan these two mushroom species are sold openly as dried mushrooms or dried mushroom "powder" on the internet and in "smoke shops" [186].

Description of the Syndrome
The syndrome produced by consuming mushrooms containing ibotenic acid and muscimol is called pantherina syndrome (or myco-atropine syndrome) [156]. The syndrome is characterized by a short latency period (30 min to 3 h) [156]. The first perceptible effects after ingestion are mainly nausea, vomiting, and diarrhea, followed by characteristic symptoms of central nervous system dysfunction (confusion, dizziness, myoclonus, visual and auditory hypersensitivity, and distortion of time and space) accompanied by mydriasis, fatigue, and drowsiness (cf. Table 5). The phenomenon of hallucinations has been discussed. After 2 h the subject presents altered states of consciousness lasting approximately 8 h [153].
Pantherina syndrome is sometimes confused with drunkenness.

Human Poisoning Cases Reported
The consumption of Amanita muscaria is connected with mysticism since the mushroom's psychotropic properties have been known and prized for several thousand years. A. muscaria was traditionally used in religious, spiritual, or shamanic rituals by some tribes in Northern Europe and Northern Asia (Siberian shamans of tribes such as the Ostyak, Vogul, Kamchadal, Koryak, and Chukchi) [153]. The "Rig Veda", the ancient Hindu text considered one of the world's great religious works (composition estimated between 1500 and 900 BC) [194], advocates "Soma". The term Soma has several meanings in Hindu mythology: a ritual drink, the plant (or the mushroom), and the god. Several hypotheses argue that Soma was extracted from Amanita muscaria [195,196]. In his book "Amanita muscaria; Herb of Immortality" Teeter considers the fly agaric to be at the centre of all religions and beliefs [197]. Theories about A. muscaria as soma have been very thoroughly debunked [198].
A. muscaria or A. pantherina poisonings can happen accidentally, through confusion with an edible mushroom species or ignorance of the fungi kingdom. However, a large proportion of these poisonings are from voluntary recreational consumption from those seeking psychotropic effects. Table 5 lists some examples. Only one case of death of a 55-year-old man attributed to an Amanita muscaria poisoning was reported [155]. Unfortunately, in this case, only muscarine in urine was quantified, neither ibotenic acid nor muscimol.

Analytical Aspect
Analytical techniques have been developed since the early 1980s with the aim of identifying and quantifying the principal mycotoxins responsible for pantherina syndrome. Liquid chromatography is the most widely used technique. It was not until the late 2000s that researchers considered the detection of isoxazoles in biological matrices (urine and serum; Table 7). Some poisoning cases have been documented where patients' biological samples were investigated for ibotenic acid and muscimol. Stříbrný et al. [176] reported varying concentrations of ibotenic acid between 32 and 55 mg/L, and of muscimol between 6 and 10 mg/L in urine (3-8 h after ingestion). Hasegawa et al. [177] reported concentrations of 96 µg/L of ibotenic acid and 101 µg/L of muscimol in the serum of a subject poisoned by A. ibotengutake (without specifying the period between ingestion and sampling).

Toxic Compounds
In 1885, Boehm and Külz isolated an oily substance from the false morel, which they believed to be the substance responsible for the mushroom's toxicity. More advanced studies have shown that it is actually a mixture of non-toxic organic acids. Gyromitrin was finally isolated, synthesized and definitively identified in 1968 by List and Luft as acetaldehyde N-methyl-N-formylhydrazone or gyromitrin (C 4 H 8 N 2 O, M = 100.1) [206][207][208]. The hydrolytic cleavage of gyromitrin ( Figure 10) leads to the formation of N-methyl-N-formylhydrazine and then methylhydrazine (or monomethylhydrazine, MMH) [209,210], which is used in astronautics as a rocket propellant [209]. Gyromitrin belongs to the hydrazine family and is volatile, thermosensitive, and very soluble in water [207]. This mycotoxin can be partially eliminated by drying or boiling the mushroom. Pyysalo [211] has shown that these measures can reduce the quantity of gyromitrin originally contained in the mushroom by up to 99-100%.

Toxic Compounds
In 1885, Boehm and Külz isolated an oily substance from the false morel, which they believed to be the substance responsible for the mushroom's toxicity. More advanced studies have shown that it is actually a mixture of non-toxic organic acids. Gyromitrin was finally isolated, synthesized and definitively identified in 1968 by List and Luft as acetaldehyde N-methyl-N-formylhydrazone or gyromitrin (C4H8N2O, M = 100.1) [206][207][208]. The hydrolytic cleavage of gyromitrin ( Figure 10) leads to the formation of N-methyl-N-formylhydrazine and then methylhydrazine (or monomethylhydrazine, MMH) [209,210], which is used in astronautics as a rocket propellant [209]. Gyromitrin belongs to the hydrazine family and is volatile, thermosensitive, and very soluble in water [207]. This mycotoxin can be partially eliminated by drying or boiling the mushroom. Pyysalo [211] has shown that these measures can reduce the quantity of gyromitrin originally contained in the mushroom by up to 99-100%.

Toxic Mechanism and Toxicity in Humans and/or Animals
Gyromitrin is classed as a GABA-inhibiting mycotoxin, group 4A in the White et al. classification [5]. Its mechanism of toxic action is connected with the production of MMH. MMH interacts with pyridoxine dependent coenzymes, resulting in inhibition of glutamic acid decarboxylase and thus reduced GABA production, causing the neurological symptoms to occur. MMH can also cause methemoglobinemia [207,212]. In addition, MMH produces radical species that lead secondarily to hepatic cytolysis [207].
N-methyl-N-formylhydrazone and methylhydrazine are known to be hepatotoxic through the mechanism of producing radical species, but they are also known to be carcinogenic in animals [209,213].
Several studies have been conducted on animals to determine the lethal dose of 50% for gyromitrin and MMH. Patocka et al. [209] reported an oral LD50 for gyromitrin of 344 mg/kg in mice, 320 mg/kg in rats, 50-70 mg/kg in rabbits, and a resistance of over 400 mg/kg in chickens. In humans, the oral LD50 is estimated at 20-50 mg/kg in adults and 10-30 mg/kg in children [207]. Studies of the lethal dose of monomethylhydrazine have also been published, reporting a dose of 4.8-8 mg/kg in adults and 1.6-4.8 mg/kg in children [212]. Pyysalo et al. reported a concentration of 50 mg of gyromitrin/kg in fresh mushrooms (Finnish species).
There is considerable variation in individual responses to gyromitrin poisoning: ranging from simple stomach upset to the death of the patient (cf. Table 8). The outcome is fatal in approximately 10% of cases [207].

Toxic Mechanism and Toxicity in Humans and/or Animals
Gyromitrin is classed as a GABA-inhibiting mycotoxin, group 4A in the White et al. classification [5]. Its mechanism of toxic action is connected with the production of MMH. MMH interacts with pyridoxine dependent coenzymes, resulting in inhibition of glutamic acid decarboxylase and thus reduced GABA production, causing the neurological symptoms to occur. MMH can also cause methemoglobinemia [207,212]. In addition, MMH produces radical species that lead secondarily to hepatic cytolysis [207].
N-methyl-N-formylhydrazone and methylhydrazine are known to be hepatotoxic through the mechanism of producing radical species, but they are also known to be carcinogenic in animals [209,213].
Several studies have been conducted on animals to determine the lethal dose of 50% for gyromitrin and MMH. Patocka et al. [209] reported an oral LD 50 for gyromitrin of 344 mg/kg in mice, 320 mg/kg in rats, 50-70 mg/kg in rabbits, and a resistance of over 400 mg/kg in chickens. In humans, the oral LD 50 is estimated at 20-50 mg/kg in adults and 10-30 mg/kg in children [207]. Studies of the lethal dose of monomethylhydrazine have also been published, reporting a dose of 4.8-8 mg/kg in adults and 1.6-4.8 mg/kg in children [212]. Pyysalo et al. reported a concentration of 50 mg of gyromitrin/kg in fresh mushrooms (Finnish species).
There is considerable variation in individual responses to gyromitrin poisoning: ranging from simple stomach upset to the death of the patient (cf. Table 8). The outcome is fatal in approximately 10% of cases [207].
Treatment of gyromitrin poisoning is symptomatic. It may include administration of vitamin B6 (pyridoxine) to stop seizures and/or anticonvulsants such as clonazepam [207,212].

Toxic Species
Gyromitrin is the main toxin in mushrooms of the genus Gyromitra of the family Discinaceae. The most common mushroom is Gyromitra esculenta (Figure 11), which is often confused with morel, hence its nickname: false morel [207] shares a subgroup with G. fastigiate [207] and G. ambigua [217]. There is no evidence that G. gigas contains gyromitrin. It would appear that a large proportion of the genus Gyromitra contains gyromitrin [209]. Treatment of gyromitrin poisoning is symptomatic. It may include administration of vitamin B6 (pyridoxine) to stop seizures and/or anticonvulsants such as clonazepam [207,212].

Toxic Species
Gyromitrin is the main toxin in mushrooms of the genus Gyromitra of the family Discinaceae. The most common mushroom is Gyromitra esculenta (Figure 11), which is often confused with morel, hence its nickname: false morel [207] shares a subgroup with G. fastigiate [207] and G. ambigua [217]. There is no evidence that G. gigas contains gyromitrin. It would appear that a large proportion of the genus Gyromitra contains gyromitrin [209]. It should be noted that G. esculenta contains other toxins beside gyromitrin: pentanal N-methyl-N-formylhydrazone, 3-methylbutanal N-methyl-N-formylhydrazone, and hexanal N-methyl-Nformylhydrazone [210]. All these compounds lead to the formation of methylhydrazine by hydrolysis [209,210]. In addition, there is a small amount of N-methyl-N-formylhydrazine in the mushroom, formed by hydrolytic cleavage [209].
This fungi genus is found mainly in the northern hemisphere (Canada, United States, and Eastern Europe). Long considered edible, G. esculenta has been the cause of many deaths.

Description of the Syndrome
The syndrome resulting from gyromitrin poisoning is called gyromitra syndrome [156]. It is characterized by a long latency period (between 5 and 12 h) after consuming the mushrooms [207]. Like the majority of mushroom poisonings, the first perceptible symptoms are nausea, vomiting, stomach pains, and sometimes bloody diarrhea, resulting in dehydration and headaches. MMH being hepatotoxic, there is often jaundice, indicating liver damage. In severe cases of poisoning there are altered states of consciousness, lack of motor coordination, seizures, and coma, which may lead to the death of the patient (c.f. Table 8).
In most cases the symptoms disappear 2-6 h after ingesting the mushrooms [212]. It should be noted that G. esculenta contains other toxins beside gyromitrin: pentanal N-methyl-N-formylhydrazone, 3-methylbutanal N-methyl-N-formylhydrazone, and hexanal N-methyl-N-formylhydrazone [210]. All these compounds lead to the formation of methylhydrazine by hydrolysis [209,210]. In addition, there is a small amount of N-methyl-N-formylhydrazine in the mushroom, formed by hydrolytic cleavage [209].
This fungi genus is found mainly in the northern hemisphere (Canada, United States, and Eastern Europe). Long considered edible, G. esculenta has been the cause of many deaths.

Description of the Syndrome
The syndrome resulting from gyromitrin poisoning is called gyromitra syndrome [156]. It is characterized by a long latency period (between 5 and 12 h) after consuming the mushrooms [207]. Like the majority of mushroom poisonings, the first perceptible symptoms are nausea, vomiting, stomach pains, and sometimes bloody diarrhea, resulting in dehydration and headaches. MMH being hepatotoxic, there is often jaundice, indicating liver damage. In severe cases of poisoning there are altered states of consciousness, lack of motor coordination, seizures, and coma, which may lead to the death of the patient (c.f. Table 8).
In most cases the symptoms disappear 2-6 h after ingesting the mushrooms [212].

Human Poisoning Cases Reported
The first cases of gyromitrin poisoning were reported in 1782, then towards the end of the 1800s [215,216]. Franke et al. [215] reported a large number of poisonings in Eastern Europe between

Conclusions
This review of the literature took an analytical perspective, and focused on highly toxic mycotoxins (orellanine, αand β-amanitin, muscarine, ibotenic acid, muscimol, and gyromitrin). It identifies a set of knowledge gaps. There is indeed a lack of scientific data, particularly regarding the metabolism of mycotoxins in biological matrices, but there is also a lack of analytical tools. There is a real need for the development and validation of specialized analytical methods adapted for the analysis of these mycotoxins in various matrices. Their implementation in the context of a clinico-biological study comparing the results of biological samples analysis (identification and assay) with the case history and clinical signs of confirmed or suspected poisoning victims could strengthen our understanding and treatment of these poisonings.