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A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Publication . Garcia, Juliana; Costa, Vera M.; Carvalho, Alexandra T.P.; Silvestre, Ricardo; Duarte, José A.; Dourado, Daniel F.A.R.; Arbo, Marcelo D.; Baltazar, Teresa; Dinis-Oliveira, Ricardo Jorge; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

2015Journal article

Open access

Amanita phalloides poisoning: Mechanisms of toxicity and treatment

Publication . Garcia, Juliana; Costa, Vera M.; Carvalho, Alexandra T.P.; Baptista, Paula; Pinho, P. Guedes de; Bastos, Maria de Lourdes; Carvalho, Félix

Amanita phalloides, also known as 'death cap', is one of the most poisonous mushrooms, being involved in the majority of human fatal cases of mushroom poisoning worldwide. This species contains three main groups of toxins: amatoxins, phallotoxins, and virotoxins. From these, amatoxins, especially α-amanitin, are the main responsible for the toxic effects in humans. It is recognized that α-amanitin inhibits RNA polymerase II, causing protein deficit and ultimately cell death, although other mechanisms are thought to be involved. The liver is the main target organ of toxicity, but other organs are also affected, especially the kidneys. Intoxication symptoms usually appear after a latent period and may include gastrointestinal disorders followed by jaundice, seizures, and coma, culminating in death. Therapy consists in supportive measures, gastric decontamination, drug therapy and, ultimately, liver transplantation if clinical condition worsens. The discovery of an effective antidote is still a major unsolved issue. The present paper examines the clinical toxicology of A. phalloides, providing the currently available information on the mechanisms of toxicityinvolved and on the current knowledge on the treatment prescribed against this type of mushrooms. Antidotal perspectives will be raised as to set the pace to new and improved therapy against these mushrooms.

2015Journal article

Open access

Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection

Publication . Garcia, Juliana; Costa, Vera M.; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifuga tion at 16,000. ×g, 4. °C, for 20. min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50. mM citric acid, and 0.46. mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22. min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%.The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.

2015Journal article

Open access

Co-ingestion of amatoxins and isoxazoles-containing mushrooms and successful treatment: A case report

Publication . Garcia, Juliana; Costa, Vera M.; Costa, Ana Elisa; Andrade, Sérgio; Carneiro, Ana Cristina; Conceição, Filipe; Paiva, José Artur; Pinho, P. Guedes de; Baptista, Paula; Bastos, Maria de Lourdes; Carvalho, Félix

Mushroom poisonings occur when ingestion of wild mushrooms containing toxins takes place, placing the consumers at life-threatening risk. In the present case report, an unusual multiple poisoning with isoxazoles- and amatoxins-containing mushrooms in a context of altered mental state and poorly controlled hypertension is presented. A 68-year-old female was presented to São João hospital (Portugal) with complaints of extreme dizziness, hallucinations, vertigo and imbalance, 3 h after consuming a stew of wild mushrooms. The first observations revealed altered mental state and elevated blood pressure. The examination of cooked mushroom fragments allowed a preliminary identification of Amanita pantherina. Gas chromatography-mass spectrometry (GC-MS) showed the presence of muscimol in urine. Moreover, through high-performance liquid chromatography-ultraviolet detection (HPLC-UV) analysis of the gastric juice, the presence of α-amanitin was found, showing that amatoxins-containing mushrooms were also included in the stew. After 4 days of supportive treatment, activated charcoal, silybin and N-acetylcysteine, the patient recovered being discharged 10 days post-ingestion with no organ complications. The prompt and appropriate therapy protocol for life-threatening amatoxins toxicity probably saved the patient's life as oral absorption was decreased and also supportive care was immediately started.

2015Journal article

Open access