I. INTRODUCCIÓN
1.1. Realidad Problemática
Amatoxins, extremely (deadly) poisonous protoplasmic toxins of some Amanita species, consist of at least nine similar bicyclic octapeptides shown in Fig. 5.1: α-amanitin, β-amanitin,
CH C NH
Fig. 5.1 Structures of the amatoxins.
C
Fig. 5.2 Structures of the phallotoxins.
γ-amanitin, ε-amanitin, amanullinic acid, amanin, amaninamid, amanullin and proamanullin.
Amatoxins occur often together with hepatotoxic phallotoxins, another group of bicyclic peptides with seven amino acids in the ring (see Fig. 5.2). They are represented mainly by phalloidin, other six actin-binding compounds identified until now are prophalloin, phalloin, phallisin, phallicidin, phallacin and phallisacin.
Virotoxins, represent the third, minor group of cyclic peptides similar to phallotoxins. This group consists of six monocyclic heptapeptides. The structures of viroidin, desoxyviroidin, [Ala]viroidin, [Ala]deoxyoviroidin, viroisin and desoxoviroisin are shown in Fig. 5.3 (Vetter, 1998).
5.2.1.2 Methods of analysis
Since botanical identification of the fungus that was eaten is in many poisoning cases impos-sible, diagnosis and its management is now based on various laboratory methods typically radioimunoassays (RIA) that were developed for examination of amatoxins (mainly α- and β-anmanitin) in serum, urine or gastrointestinal fluids. The test kits use either 3H tracer or 125 I tracer. The sensitivity of commercial RIA assays is around 0.1 ng mL−1for serum and 0.25 ng mL−1for urine (Gonmori and Yoshioka, 2003).
As an alternative, sensitive HPLC methods employing conventional detectors are available (Gonmori and Yoshioka, 2003). Also, the possibility to use of capillary zone electrophoresis (CZE) with photodiode array detection has been demonstrated (Br¨uggemannet al., 1996).
Nowadays, combined liquid chromatography mass spectrometry (LC/MS) is a prominent technique for the comprehensive analysis of amatoxins (and many other mushroom toxins), both inAmanita fruiting bodies and biological fluids (Drummer, 1999; Maurer et al., 1998, 2000; Zhanget al., 2005). It should be noted, however, that LC/MS availability for routine use is still limited, actually, most clinical laboratories do not use even the older RIA technique,
N Fig. 5.3 Structures of the virotoxins.
hence the diagnosis based entirely on symptomology and recent dietary history is still common is many places.
5.2.1.3 Incidence and levels of occurrence
Amanita phalloides, the most poisonous known mushroom (so-called green Death Cap), is commonly found all over Europe, North America and some other areas with mild climates. It may contain up to 80 mg kg−1of fresh tissue α-amanitin and 50 mg kg−1β-amanitin. These compounds account in most cases for more than 90% of total amatoxins. In addition to them, phalloidin is present, its content may approach 100 mg kg−1. Phalloidin is also found in the edible (and sought after) Blusher (Amanita rubescens) (Litten, 1975).
Occasionally, a closely related sub-speciesAmanita phalloides var. verna (in the older pa-pers misclassified as separate species,A. verna), can be found. Its cup instead of olive green is pale or even white and the content of amatoxins may be lower compared to green cup.
Another white species,A. virosa (the Destroying Angel), differs from the above poisonous mushrooms in its toxin pattern: α-amanitin may be completely replaced by amaninamide.
The overall concentration of amatoxins inA. virosa varies in the range 40–200 mg kg−1 fresh tissue (Faulstich, 2005). In this species, also virotoxins are found exclusively. There are several other fungi genera, such asGalerina and Lepiota, containing amatoxins, nevertheless, their content is several times lower as compared to previous highly poisonous representa-tives ofAmanitus genus. The distribution of toxins in various tissues of Amanita species was studied by several groups. In an older study (Enjalbertet al., 1993) Amanita phalloides mush-rooms representing three specimens at two carpophore development stages were examined.
Substantial differences in the tissue toxin content were found. The ring displayed a very high level of toxins, whereas the bulb had the lowest toxin content. Compositional differences in relation to the nature of the tissue were also noted. The highest amatoxin content was found in the ring, gills and cap, whereas the bulb and volva were the richest in phallotoxins.
Furthermore, variability in the toxin composition was observed, it is assumed that the differ-ences in the distribution of individual toxins in the tissues might be related to the carpophore developmental stage. Generally, young fruit body contains lower, and the well-developed fun-gus higher concentrations of toxins, but their concentrations in certain species are variable, even among mushrooms collected in the same region (Vetter, 1998). The follow-up study (Enjalbertet al., 1999) conducted on 25 Amanita phalloides carpophores collected from three sites in France that differed in their geological and pedological characteristics confirmed the above conclusions.
5.2.1.4 Exposure assessment
Often the cause of poisoning by these deadlyAmanitas is, akin to case of other mushrooms, i.e. their mistaking for edible species. In this respect white-coloured species are the most dangerous since they are erroneously considered to be edibleMacrolepiota or Agaricus. It should be emphasized that amatoxins peptides are not destroyed by cooking and can be kept for years if dried, they will only decompose slowly when exposed to ultraviolet (UV) light for several months. Regarding detoxification by organisms, unfortunately, no protease known would cleave the peptide bonds in the cyclic peptide. In other words, the consumer’s exposure correlates with amount of fresh mushrooms consumed and their content of toxins.
The LOD50of major amatoxins inAmanita phalloides, α-amanitin and β-amanitin, was in experiments with mice estimated in the range 0.3–0.6 mg kg−1of body weight. Given the estimated lethal dose for humans (0.1 mg kg−1body weight), a full-grown mushroom (25 g) will be sufficient to kill a human (Faulstich, 2005).
The major toxic mechanism associated with amatoxins poisoning is the inhibition of RNA polymerase II, a vital enzyme in cell metabolism. In this way, mRNA synthesis is affected (amatoxins inhibit protein biosynthesis at the transcriptional level), leading to cell death.
The liver is the principal organ affected, as it is the organ which is first encountered after absorption in the gastrointestinal symptom, though other organs, especially the kidneys, are susceptible.
The course of amanitin intoxication has three chronological phases. A latent phase takes approximately 6–24 hours and rarely exceeds 48 hours. A gastrointestinal phase lasts typically 2–3 days and is associated with abdominal pain, vomiting and diarrhoea causing dehydration, hypovolaemia, electrolyte and acid–base disorders. The third phase, so called hepatic, begins 36–48 hours after ingestion. Hepatitis becomes clinically evident with the onset of jaundice on the 3rd–4th day after ingestion; hepatic coma, bleeding and anuria may occur in intox-icated patients. When liver damage is reversible, patients usually make a slow and steady recovery. In fatal cases, death occurs within 6–16 days. In spite of improved knowledge of amatoxins poisoning, fatalities are still relatively high (according to some estimates up to 30%) since there is no specific therapy available (Faulstich and Wieland, 1992; Karlson-Stiber and Persson, 2003). In total, more than 90% of all fatal cases of mushroom poisoning in Europe are due toAmanita phalloides.
Compared to amatoxins, phallotoxins are highly toxic to liver cells (Wieland and Govin-dan, 1974), their intoxication mechanism is believed to be due to the specific binding of the toxin to F-actin, which subsequently inhibits the depolymerization of F-actin into G-actin.
However, since their absorption from the gastrointestinal tract is not significant, they do not seem to play a major role in human toxicity. Virotoxins are closely related to phallotoxins with respect to their structure and toxicity, but they do not exert any acute toxicity after ingestion in humans (Karlson-Stiber and Persson, 2003).
5.2.2 Orellanine
5.2.2.1 Occurrence route or mechanism of formation
Orellanine is a nefrotoxin exclusively occurring in mushrooms representing theCortinarius genus. The chemical composition of orellanine remained unknown until mid of the 1970s when it was identified as a hydroxylated bipyridyl-N, N-dioxide (Oubrahimet al., 1998). In the most stable form of orellanine, the nitrogen atoms are positively charged. It is supposed, that reactive semi-quinone, see Fig. 5.4, is produced in cells by a peroxidase reaction. The semi-quinone is a radical that probably causes intracellular depletion of glutathione and ascorbate as the toxic event. Orellanine is inCortinarius mushrooms typically accompanied by the corresponding monooxide, orellinine, which is less toxic. When irradiated by UV light, non-toxic stable orelline is formed through the loss of N-oxides (Ruedlet al., 1989).
5.2.2.2 Methods of analysis
Several analytical methods were developed both for the analysis of mushrooms and biological samples, such as plasma and renal tissue. In the later case, TLC can be used for separation prior to proof of the presence of toxins under UV light. Orellanine is visible as navy blue, orellinine as dark blue, and orelline as light blue fluorescent spots on silica TLC plates (Hornet al., 1997). Quantitative analysis of orellanine in plasma samples, or in (rat) urine samples, was performed by two-dimensional TLC on cellulose employing spectrophotometric evaluation of orelline produced by UV-induced decomposition of orellanine. HPLC was used for the analysis of orellanine in mushroom extracts (Faulstich, 2005). Very simple and quick methods for the identification of orellanine in mushroom isolates based in TLC and electrophoresis was developed by Oubrahimet al. (1997).
5.2.2.3 Incidence and levels of occurrence
The orellanine levels and its tissue distribution were studied inCortinarius orellanus and Cortinarius rubellus species (Koller et al., 2002). The analysis of caps showed the content of toxin to be (expressed on dry weight basis) 9400 mg kg−1 and 7800 mg kg−1in stems 4800 mg kg−1and 4200 mg kg−1, and in spores 3100 mg kg−1and 900 mg kg−1, respectively.
In mycorrhiza roots fromC. rubellus, the orellanine content was 0.03%. In another study (Faulstich, 2005), the amount of orellanine was determined as ca. 14 000 mg kg−1dry weight inC. orellanus, and 9000 mg kg−1dry weight inC. speciocissimus.
+ +
Fig. 5.4 Structures of orellanine and its degradation products orellinine, orelline and radical semiquinone of orellanine.
5.2.2.4 Exposure assessment
Bipyridines with positively charged nitrogen atoms were already known to be poisonous be-fore the structure of orellanine was elucidated. At the molecular level, orellanine was shown to be an inhibitor of alkaline phosphatase (Ruedlet al., 1989). Symptoms of orellanine poi-soning are typical of renal damage, developing over several days, in some cases up to 2 weeks after the mushroom meal (Danelet al., 2001). During the latent period, mild gastrointesti-nal disorders occur, which may be overlooked. Accordingly, patients present themselves at hospital only at the stage when renal failure has developed. In this respect, orellanin is par-ticularly insidious. Lethal doses of orellanine are known for the mouse only, corresponding to 15–20 mg kg−1body weight for intraperitoneal and 33–90 mg kg−1body weight for oral administration (Faulstich, 2005).
5.2.3 Muscarine
5.2.3.1 Occurrence route or mechanism of formation
L-(+)-Muscarine, [(4R)-4-hydroxy-5-methyl-oxolan-2-yl]methyl-trimethyl-azanium (Fig.
5.5) is a toxic chiral quaternary amine occurring in mainly mushrooms ofClitocybe and Inocybe genera. Traces of three other stereoisomers (theoretically may exist 8 isomeric sub-stances), epimuskarin, epiallomuskatin and allomuskarin (Fig. 5.5) were also detected in these toxic fungi, nevertheless, they possess only low biological activity.
5.2.3.2 Methods of analysis
Muscarine can be detected by high performance thin layer chromatography (HPTLC). Using Dragendorff reagent, muscarine appears as an orange spot on the plate (Stijve, 1981).
Recently, Wai-cheunget al. (2007) developed a new multi-detection method using HPLC separation of mushroom toxins employing an HILIC column with amide-based stationary phase that enables hydrophilic interactions and tandem mass spectrometry (MS-MS) with electrospray ionization (ESI+) for high sensitive detection. This LC/MS-MS method was successfully employed to simultaneously separate several polar mushroom toxins, including amanitins and phallotoxins.
5.2.3.3 Incidence and levels of occurrence
Muscarine is present in all the parts of respective most poisonous genera,Inocybe and Clito-cybe. In a specimen like I. patouillardi, I. fastigiata, I. geophylla and C. dealbata, it accounts for 1000–3000 mg kg−1of dry weight. WhileInocybe mushrooms are mycorrhizal on conifers or broad-leafed trees,Clitocybe mushrooms are saprophytic and grow on forest litter or grassland humus. Both these genera occur commonly in summer and autumn and have a worldwide distribution.
+
N(CH3)3 H3C
HO
O H3C O N(CH3)3
HO
+
O H3C
HO
N(CH3)3
+
O
H3C N(CH3)3 HO
+
Fig. 5.5 Structures of muscarine, epimuscarine, epiallomuscarine and allomuscarine.
Small amounts of muscarine, at a maximum of 90 mg kg−1, are also present inA. mus-caria. Although this species got its name due to the presence of muscarine (this toxin was for the first time isolated and identified), the main toxic principle in it is muscimol and its pre-cursor ibotenic acid (Faulstich, 2005). Harmless levels of muscarine, typically not exceeding 20 mg kg−1, occur in several other genera, for instance Amanita, Boletus, Hygrocybe, Lactarius, Mycena and Russula.
5.2.3.4 Exposure assessment
Due to certain structural similarity, muscarine mimics the action of the neurotransmitter acetylcholine at metabotropic receptors that are also known under the name muscarinic acetylcholine receptors. It should be noted that muscarine is not destroyed by heating during cooking (Lambertet al., 2000). So when a mushroom meal with a high muscarine content is ingested, poisoning characterized by profuse sweating, perspiration, salivation, urination, gastric upset, emesis and lachrymation occurs, and vomiting may occur. Symptoms onset is soon, typically within 15–30 minutes after ingestion of the mushroom. With large doses, these symptoms may be followed by abdominal pain, severe nausea, diarrhoea, blurred vi-sion and laboured breathing. Intoxication generally subsides within 2 hours. The species such asAmanta muscaria containing less muscarine are only occasionally responsible for cholinomimetic signs. Generally, fatalities due to muscarine are rare, and are mostly limited to victims with pre-existing health problems.