Amanita phalloides POISONING: MECHANISMS OF TOXICITY AND TREATMENT

Amanita phalloides POISONING: MECHANISMS OF TOXICITY AND TREATMENT

                    By :

                                                      Juragan Tugas Kuliah

                   

 STRUCTURAL ASSIGNMENT OF BIOLOGY OF MACROSCOPIC FUNGI

MINISTRY OF RESEARCH, TECHNOLOGY,  AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY

FACULTY OF BIOLOGY

PURWOKERTO

2017

FOREWORD

Praise gratitude authors pray to Allah SWT who has bestowed His grace and guidance, so that authors can finish this paper. The content of this paper is about Poisonous mushroom. This paper is written in order to fulfill the structural assignment in the course of Biology of Macroscopic Fungi on Biology Faculty of Jenderal Soedirman University, Purwokerto.

The preparation of this paper is inseparable from the involvement of various parties, therefore the authors convey the thanks to Drs.Aris Mumpuni, M.Phil., Dr. Nuniek Ina Ratnaningtyas, ..... as the lecturers in the course of Biology of Macroscopic of Class D International.

The authors hope later this paper can be useful in adding good science, especially the development of mushroom science and its application.

                                                                        Purwokerto,  6^th of December 2017   

Author

Introduction

            Mushrooms are one of the most interesting and striking natural inhabitants in the world, because of their highly distinctive tastes and uses. They are such a strange organism  in which sometimes have beautiful shapes and forms. Some of them are medicinal and the others are poisonous or even lethal. Poisonous mushrooms are also one of the most catchy type of mushroom, they have attracted scientists’s attention because of their remarkable morphological and physiological properties.

The public demand increasing for wild edible mushrooms have also contributed to the increasing interest in their picking and consumption, which enhances the risk of intoxications by toxic mushrooms. Garcia et al. (2015), described that although the warnings of the risks have shared to public, collectors may still be confused for both edible and toxic mushrooms, due to misidentification based on morphological characteristics. Toxic mushrooms can be grouped based on their toxic components: cyclopeptides, gyromitrin, muscarine coprine, isoxazoles, orellanine, psilocybin, and gastrointestinal irritants. By then, cyclopeptides-containing mushrooms are the most toxic species throughout the world, being responsible for 90–95% of human fatalities. The main toxic agents are amatoxins that are present in three genera: Amanita (mainly Amanita phalloides, A. virosa and A. verna); Lepiota (the most frequently reported is L. brunneoincarnata) and Galerina (the most common being Galerina marginata). Among these species, A. phalloides is the most responsible for the majority of fatal cases due to mushroom poisoning .

Amatoxin poisoning has emerged as a serious public health problem worldwide. Therefore, this review aims to provide the state of the art concerning the mechanisms of toxicity, patterns of clinical presentation and management of amatoxin poisoning, focusing on the efficacy and limitations of the most commonly used antidotes (Garcia et al., 2015).

Morphologically, A. phalloides is greenish yellow, darker in the center and faintly streaked radially. It has smooth moist cap, which is 6–12.5 cm across and easily peeled. The stalk is smooth, white and 6–12.5 cm high. There is an irregular ring near the top of the stalk and a bulbous cup at the base. The fruiting body emanates a sweetish and not unpleasant smell. Its taste is pleasant, according to the survivors after intoxication.  A. phalloides is distinguished from other species, like Volvariella volvacea, by their irregular ring near the top of the stalk, the bulbous cup at the base and white gills under the cap that are not attached to the stem. The morphology of the bulbous cup has been an important feature to distinguish Amanita from other resembling genera. However, inexperienced collectors break the specimen off at the stem destroying or neglecting some of these characteristics, which puts the consumers at risk of intoxication. Moreover, non-Amanita containing-amatoxins species exist placing more people at risk. Additionally, mushroom species have mutable appearances at different times of year and at different locations, depending on weather, soil, and time of harvest, which makes more challenging the correct mushroom identification for collectors (Garcia et al., 2015). Parnmen et al. (2016) listed several types of toxins which are responsible for mushroom poisoning, there are cyclopeptide, orellanine, monomethyl hydrazine, disulfiram-like, hallucinogenic indole, muscarinic, isoxazole, and gastrointestinal specific irritants.

A. phalloides is the predominant European poisonous mushroom, particularly in Central and Occidental Europe. Several cases of A. phalloides poisoning have also been reported in northeastern United States, Central and South America, Asia, Australia. This species is an ectomycorrhizal fungus that forms symbiotic relationships with a variety of tree species, such as beech, oak, chestnut, and pine. The best seasons of the year for A. phalloides fructification are spring, late summer, and autumn, and therefore the majority of the intoxication cases occur in those seasons (Garcia et al., 2015).

The distribution of the toxins through the carpophore is unequal in the body parts of this mushroom. The highest amatoxins content was found in the ring, gills and cap, while the volva had the richest in the amount of phallotoxins. The quantity of toxins on the carpophore elements is affected by the collection site and the age of the collected species. The collection site (mainly soil characteristics) determines toxins’ composition of each mushroom, mostly the predominance of either acidic or neutral phallotoxins. Regarding the maturation state, the content of amatoxins is relatively high during the early development stages (button, button with broken outer veil, and pileus revealed from outer veil) and decreases in the mature (completely developed fruit body with convex cap) and old (wilted fruit body with reflexed cap) stages (Enjalbert et al., 1996).

Discussion

According to Morel et al. (2016), the frequency of mushroom fatal poisonings recorded in emergency medicine units increases worldwide. Over 90% of human casualties are caused by the ingestion of amatoxin-containing species of the genus Amanita, mainly Amanita phalloides. Garcia et al. (2015) stated that A. phalloides contains three classes of cyclic peptide toxins, it can be grouped into amatoxins, phallotoxins, and virotoxins. All groups of toxins contain a tryptophan residue substituted at position 2 of the indol ring by a sulfur atom. They have distinct toxicological profiles: amatoxins are highly toxic, whereas phallotoxins and virotoxins are less toxic  but act quickly, causing death within 2–5 h.

Amatoxins are a group of nine bicyclic octapeptides (with an indole-(R)-sulphoxide bridge) resistant to heat, freezing, drying and digestion. They are absorbed in the gastro-intestinal tract and are considered as the agent responsible for poisoning (Morel et al., 2016). The main toxicological studies were focused on α-amanitin and β- amanitin toxins, thus no final conclusions can be drawn regarding the potential differences between neutral and acid amatoxins. Amatoxins only differ by the number of hydroxyl groups and by an amide carboxyl exchange. These toxins have great heat stability and this property combined with their solubility in water make them exceptionally toxic as they are not destroyed by cooking or drying (Garcia et al., 2015).

 Another toxin of this mushroom according to Morel et al. (2016) is phallotoxin. Phallotoxins are a group of seven bicyclic heptapeptides (with an indole-thio-ether bridge) unstable to heat. They won’t be absorbed in the gastrointestinal tract and are therefore not considered to be responsible for poisoning. Garcia et al. (2015) stated that Phallatoxin is formed by at least seven different compounds: phalloidin, phalloin, prophallin, phallisin, phallacin, phallacidin, and phallisacin. From these, phalloidin, phalloin, prophallin, and phallisin are classified as neutral phallotoxins, whereas phallacin, phallacidin, and phallisacin are acidic phallotoxins.

Virotoxins are monocyclic peptides formed by at least five different compounds: alaviroidin, viroisin, deoxoviroisin, viroidin, and deoxoviroidin. The structure and biological activity of virotoxins are similar to that of phallotoxins, thus suggesting that virotoxins are biosynthetically derived from phallotoxins or share common precursor pathways. As with phallotoxins, virotoxins are not considered to have significant toxic effects after oral exposure. At the molecular level, like phallotoxins, they interact with actin, stabilizing the bonds between actin monomers and preventing microfilaments depolymerization. However, the ultraviolet-spectra of interaction between actin and virotoxins is different from that of actinphallotoxins, suggesting a different molecular interaction (Garcia et al., 2015). Therefore Amanita fatal poisonings are associated with amatoxins (α-, β- and γ-amanitins accounting for 40% of the amatoxin content) which are considered as one of the most violent natural poisons. Amatoxins are absorbed in the intestinal tract and follow the enterohepatic cycle thereby increasing toxins half-life and intoxication severity. Toxins accumulate in the liver. Excretion is mainly urinary. Acute tubular necrosis may occur in the kidney after ingestion of amatoxin-containing mushrooms (Morel et al., 2016).

            Amatoxins do not undergo metabolism and they are excreted in large quantities in the urine during the first days following ingestion, with maximal excretion occurring in the first 72 h. A small amount can be eliminated in bile and may be reabsorbed via the enterohepatic circulation, which prolongs the body burden to these toxins. Intestinal elimination also seems to occur. In a human intoxication report 6.3 mg of α-amanitin was eliminated in the feces over a period of 24 h; this amount is believed to be lethal in an adult. There are three distinct phases of the A. phalloides toxic syndrome have been established in the literature: 1) gastrointestinal phase, 2) latent period and 3) the hepatorenal phase. The first stage of A. phalloides syndrome occurs abruptly, 6– 24 h after ingestion, and is characterized by nausea, vomiting, diarrhea (occasionally bloody), abdominal pain, and hematuria. The latent period is characterized by absence of symptoms, whilst progressive deterioration of hepatic and renal function is occurring (Becker et al., 1976). Hepatic lesions are accompanied by increased serum concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). The blood coagulation is also severely disturbed, which may give rise to internal bleeding. The pathological hallmark of amatoxin poisoning is the development of liver necrosis and this characterizes the hepatorenal phase. The patients progressively lose kidney and liver functions and may develop jaundice, hypoglycemia, oliguria, delirium, and confusion.

            The toxicity mechanism of this mushroom is varry among each individu with several toxicity mechanisms that have been attributed to amatoxins. The main mechanism seems to be their known ability to noncovalently bind and inhibit RNA polymerase II (RNAP II) activity in the nucleus. Many experimental studies have been conducted to get a better understanding of the interaction with RNAP II.

           

Figure 1. The toxicity mechanisms of A. Palloides.

            The main toxicity mechanism of α-amanitin is the inhibition of RNA polymerase II. Signaling pathways involved in α-amanitin-induced toxicity. Other mechanisms have been suggested and include the formation of reactive oxygen species (ROS) leading to oxidative stress related damage. Generation of ROS may also be induced by increase of superoxide dismutase (SOD) activity and inhibition of catalase activity. Amatoxins may act synergistically with tumor necrosis factor (TNF), to induce apoptosis, though the underlying mechanisms are not yet known. Amatoxins-induced apoptosis may also be caused by the translocation of p53 to the mitochondria causing alteration of mitochondrial membrane permeability through formation of complexes with protective proteins (Bcl-xL and Bcl-2). These changes result in the release of cytochrome c into the cytosol and activation of the intrinsic pathway of apoptosis. Question marks indicate that the mechanisms that remain unknown (Garcia et al., 2015).

            Severals studies have been conducted to limit the toxicity of amatoxins, Garcia et al. (2015) in vitro studies using human hepatocytes provided some evidence to support the effectiveness of benzylpenicillin in limiting the cytotoxicity of amatoxins. However, the effectiveness of benzylpenicillin may be species dependent, as it was not found to be effective in limiting hepatic injury. Cytotoxicity evaluation on cultured human hepatocyte using MTT reduction and leakage assays was performed after 12, 24 and 48 h exposure to α-amanitin (2 μM) and/or silybin. The treatment with silybin showed a strong protective effect against cell damage in α-amanitin-induced toxicity. The postulated protective mechanisms of action mediated by silybin are associated to its strong antioxidant activity, which could explain its action against hepatotoxic agents that act through oxidative stress. Silybin and silymarin reduce the free radical load, stimulate the activity of SOD and increase GSH levels.

            The optimal management of the A. phalloides poisoning remains to be determined. Garcia et al. (2015) stated that retrospective analysis of the applied therapy, specifically using benzylpenicillin, ceftazidime, silybin, and Nacetylcysteine, has revealed contradictory results regarding to their clinical effectiveness. Silybin seems a promising drug to prevent amatoxins-induced intoxications symptomatology demonstrating a good safety profile and so far it has presented the lowest mortality rate of the applied treatments. Even so, more clinical studies and in vivo experimental data are needed to prove its use in the clinical practice.

Conclusion

Based on the above discussion, it can be concluded that A. phalloides is one of the most toxic mushrooms and is involved in the majority of human fatal cases of mushroom poisoning. The true incidence of amatoxin poisoning is unknown due to sub notification cases of intoxication cases, and therefore mortality rates reported in the literature may be significantly underestimated. There some variations of toxicity mechanisms of this mushroom, which are the inhibition of RNA polymerase II and the formation of Reactive Oxygen Species (ROS). Untill now, silybin has known as the most promising drug to prevent amatoxins-induced intoxications symptomatology demonstrating a good safety profile.

REFERENCES

Enjalbert, F., Cassanas, G., Guinchard, C., & Chaumont, J. P. 1996. Toxin composition of Amanita phalloides tissues in relation to the collection site. Mycologia, 909-921.

Garcia, J., Costa, V. M., Carvalho, A., Baptista, P., de Pinho, P. G., de Lourdes Bastos, M., & Carvalho, F. 2015. Amanita phalloides poisoning: Mechanisms of toxicity and treatment. Food and chemical toxicology, 86, pp. 41-55. 

Morel, S., Fons, F., Rapior, S.,  Dubois, V., Vitou, M., Portet, K.,  Dore, J., &        Poucheret, P. 2016. Decision-making for the detection of amatoxin poisoning: a comparative study   of standard analytical methods. Cryptogamie, Mycologie,        37(2), pp. 217-239

Parnmen, S., Sikaphan, S., Leudang, S., Boonpratuang, T., Rangsiruji, A., &          Naksuwankul, K. 2016. Molecular identification of poisonous mushroom using        nuclear ITS region and peptide toxins: a retrospective study on fatal cases in   Thailand. The journal of toxicological sciences, 41(1), pp: 65-76.