Amatoxins are a family of cyclic peptides, with a-amanitin and b-amanitin (Figure 3.1) accounting for >90% of the total amatoxins. In A. virosa, mushrooms collected in Virginia a-amanitin found to be completely replaced by amaninamide (Figure 3.1). The peptides are not destroyed by cooking and
can be kept for years if dried, but will decompose slowly when exposed to ultraviolet (UV) light for several months. No protease known would cleave the peptide bonds in the cyclic peptide. Amatoxins inhibit protein biosynthesis at the transcriptional level. There was virtually no difference found in the toxic activities of a-amanitin, p-amanitin, and amaninamide. By tightly binding to the enzyme transcribing DNA into mRNA, the toxins inhibit the synthesis of the templates required for proteins to be synthesized in the cell. If transcription is not restored within a certain period of time, cells poisoned by amatoxins die from programmed cell death (apoptosis). Amatoxins have a poor rate of penetration through cell membranes and therefore preferentially enter cells possessing an amanitin-transporting system, as is present in cells of the intestinal mucosa and in hepatocytes.
The specificity of amatoxins for cells in the gastrointestinal (GI) tract and in the liver explains the symptoms of amatoxin poisoning. Onset of symptoms occurs at the earliest six hours, in most cases eight to twelve hours, after the mushroom meal, with vomiting, diarrhea, abdominal cramps, and nausea, known as the GI phase. A plausible explanation for the long latent period is that poisoned cells can live on their stock of mRNA for some hours until the RNAs are degraded, according to their specific life time. The latent period is so characteristic that it represents an important diagnostic tool for amanitin poisoning. After the GI phase (days one to two), patients experience a treacherous phase of recovery, but may suffer from the first coagulation disorders, such as decrease in prothrombin time. The hepatic phase (days three to four) develops with an increase of liver transaminases in serum, LDH, and bilirubin. In severe cases, acute hepatic failure will develop, associated with severe coagulation disorders and encephalopathy. Acute renal failure may occur at this stage, indicating a poor prognosis due to heavy toxin exposure. Patients eventually die after six to sixteen days, exceptionally as late as twenty days after ingestion.
There is no specific therapy for amatoxin poisoning. Intense supportive care, including rehydration, balance of electrolyte loss, substitution of blood glucose, administration of activated charcoal, and, if necessary, enhanced diuresis, should be started as soon as possible. Extracorporal purification procedures, such as hemodialysis, hemoperfusion, or plasmapheresis, are no longer recommended because of the low plasma concentration of amatoxins at the time of hopitalization; however, infusion of charcoal slurry may prevent residual amatoxins, or amatoxins excreted in bile, from being (re)absorbed. Kidney function should be controlled carefully, and slightly enhanced diuresis seems important (100 to 200ml/h). Silymarin (silibinin), and, less efficiently, penicillin, have been proved in experimental animals to intercept reabsorption of amanitin excreted by the liver due to enterohepatic circulation. If very high doses of amanitin were ingested, a fatal outcome seems avoidable only by liver transplantation, which has recently been performed successfully. Mortality rates were 30% in former decades but have now dropped to around 11%, mainly due to intense supportive care. Such values are ambiguous, however, given the lack of an obligatory classification system of severity. (For recent reviews on the management of amatoxin poisoning, see: Faulstich and Zilker, 1994; Enjalbert et al., 2002; Persson and Karlson-Stiber, in press).
It is possible to use the newspaper test to detect amatoxins in a drop of mushroom juice. In this test, amatoxins develop a purple-brownish color when concentrated hydrochloric acid is added to the dried spot due to the lignin present in the newspaper. However, several mushroom species that contain no amatoxins will give a false-positive result with this test (Seeger, 1984). Analytical procedures with much higher sensitivity for detecting amatoxins include high performance liquid chromatography (HPLC) and various radioimmunoassays. These are suited also for detecting amanitin in urine samples of patients, with a limit of detection of ca. 10 ng/ml. In the past, two amatoxin-specific radioimmunoassays were brought to market, one from the author's laboratory, the other from a Swiss group. However, interest in the analysis of urine samples has since declined because general agreement was reached that amatoxin concentration in urine is of no prognostic value for the course of an individual intoxication. Radioimmunoassays and HPLC analysis work for serum samples also, but in most cases the serum of patients is free of amatoxins at the time of hospitalization. However, such analytical procedures may still be useful for analysing mushroom remains.
Beside amatoxins, A. phalloides and A. virosa contain a second family of toxic, cyclic peptides, the phallotoxins, with phalloidin and phallacidin being the main components. There is convincing evidence that phallotoxins do not contribute to human mushroom poisoning. The same is true for the virotoxins, a family of cyclic peptides structurally related to the phallotoxins and found exclusively in A. virosa. Phallotoxins and virotoxins share the target protein polymeric actin, in non-muscle cells, preferentially in hepatocytes. Several species of Amanita, among them A. phalloides and A. virosa, but also edible species of this genus such as A. rubescens, contain so-called hemolysins, proteins with very potent cytolytic activity. These proteins are destroyed during cooking (temperature >65°C), and hence do not contribute to human poisoning.
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