Liver transcriptome analyses of acute poisoning and recovery of male ICR mice exposed to the mushroom toxin α-amanitin.

Approximately 70-90% of mushroom poisoning deaths are caused by α-amanitin-induced liver injury resulting from RNA polymerase II (RNAP II) inhibition. Liver regeneration ability may contribute greatly to individual survival after α-amanitin poisoning. However, it is unclear what cellular pathways are activated to stimulate regeneration. We conducted dose-effect and time-effect studies in mice that were intraperitoneally injected with 0.33-0.66 mg/kg α-amanitin to establish a poisoning model. The liver/body weight ratio, serological indices, and pathology were evaluated to characterize the liver injury. In the time-effect study, the liver transcriptome was analyzed to explore the mRNA changes resulting from RNAP II inhibition and the underlying pathways associated with recovery. Based on the two animal studies, we established a poisoning model with three sequential liver states: early injury, regulation, and recovery. The mRNA changes reflected by the differentially expressed genes (DEGs) in the transcriptome could be used to illustrate the inhibition of RNAP II by α-amanitin. DEGs at four key time points were well matched with the three liver states, including 8-h downregulated genes in the early injury state, 16-h and 72-h upregulated genes in the regulation state, and 96-h upregulated/downregulated genes in the recovery state. By clustering analysis, the mTOR signaling pathway was screened out as the most promising potential pathway promoting recovery. The results of our investigations of the pathways and events downstream of the mTOR pathway indicated that the activation of mTOR probably contributes crucially to liver regeneration, which could be a promising basis for drug development.

An effective antidotal combination of polymyxin B and methylprednisolone for α-amanitin intoxication.

Amanita phalloides is one of the most toxic mushrooms worldwide, and it is involved in the majority of human fatal cases of mushroom poisoning. α-Amanitin, the most deleterious toxin of A. phalloides to humans, inhibits RNA polymerase II (RNAPII), causing hepatic and renal failure. Previously, we have shown that polymyxin B (polB) reverts α-amanitin inhibition of RNAPII, although it was not able to guarantee the full survival of α-amanitin-intoxicated mice or prevent α-amanitin pro-inflammatory effects. α-Amanitin is also a substrate of the organic-anion-transporting polypeptide 1B3 (OATP1B3) and Na(+)-taurocholate cotransporter polypeptide (NTCP) transporters. Therefore, in the present work, we used a combination of polB [(2.5 mg/kg intraperitoneal (i.p.)] with the anti-inflammatory and NTCP inhibitor drug, methylprednisolone (MP) (10 mg/kg i.p.), as an attempt to fully revert α-amanitin-induced toxicity (0.33 mg/kg i.p.) in CD-1 mice. Results showed that the administration of the polB + MP combination, 4 h after α-amanitin, led to the full survival of the intoxicated animals, with a significant attenuation of α-amanitin-induced renal and hepatic necrosis. Also, the combination polB + MP led to a decrease of aminotransferase plasma levels, of the renal myeloperoxidase activity and of renal inflammatory cell infiltrate promoted by α-amanitin, although not preventing any of the hepatic pro-inflammatory effect of the toxin. The obtained results indicate that this combination may represent an important and valuable therapeutic approach to be used against α-amanitin intoxication.

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

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.

[Two different outcomes after Death Cap mushroom intoxication]. / To forskellige forløb af forgiftning med grøn fluesvamp.

Death Cap is one of the most lethal mushrooms in Denmark and may be mistaken for a non-toxic Asian mushroom. We report on two accidental cases admitted 12 and 17 hours after ingestion presenting with gastroenteritis and decline in liver function. The patient who arrived after 12 hours responded well to intensive treatment of liver failure and was discharged after 18 days. The other patient deteriorated in spite of intensive treatment and underwent liver transplantation. She was later discharged. Early diagnosis and treatment is essential.

Amanitin toxicosis in two cats with acute hepatic and renal failure.

Amanitin is a toxic cyclopeptide present in several species of poisonous mushrooms. Amanitin toxicosis was diagnosed in 2 cats from separate premises. Both cats initially had lethargy and vomiting, and they rapidly developed depression and neurological signs over 24-48 hours. Marked elevation of alanine aminotransferase was the primary finding, with subsequent serum chemistry values compatible with hepatic and renal failure. Histopathological findings consisted of submassive to massive acute hepatic necrosis, renal proximal tubular epithelial necrosis, and foci of necrosis and inflammation in the gastrointestinal tract. Amanitin exposure was confirmed postmortem by detection of α-amanitin in the kidney by liquid chromatography-mass spectrometry. A similar clinical course and pathological changes are reported in human and canine amanitin intoxication; however, gastrointestinal lesions are not typically described.

Study on genes with altered expression in alpha-amanitin poisoned mice and evaluation on antagonistic effects of traditional Chinese medicines against toxicity of alpha-amanitin.

The forward and reverse cDNA subtractive libraries before and after the toxic effect of alpha-amanitin were constructed by suppression subtractive hybridization and randomly selected clones from each subtractive library were screened by PCR and dot blot hybridization. A total of 85 genes with altered expression were finally identified, with 41 genes from the forward library and 44 from the reverse library. Subsequently, the antagonistic effects of candidate traditional Chinese medicines were evaluated based on the genetic transcription levels of the genes with significant altered expression, including Catnbeta, Flt3-L, IL-7r and Rpo2-4. The results indicated that Silybum marianum (L.) Gaert and Ganoderma lucidum had significant down-regulated effects on the transcription level of Catnbeta that was up-regulated by alpha-amanitin, and the two herbs also up-regulated the transcription levels of Flt3-L and Rpo2-4. Silybum marianum (L.) had significant up-regulated effects on the IL-7r that was down-regulated by alpha-amanitin. These preliminary studies suggested that Silybum marianum (L.) and Ganoderma lucidum were effective antagonists against the toxicity of alpha-amanitin.

Failure of benzylpenicillin, N-acetylcysteine and silibinin to reduce alpha-amanitin hepatotoxicity.

BACKGROUND: Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology. The objective of this study was a comparative evaluation of benzylpenicillin (Bp), acetylcysteine (ACC) and silibinin (Sil) efficacy as antidotes in hepatocytes intoxicated with alpha-amanitin (alpha-AMA). MATERIALS AND METHODS: All experiments were performed on cultured canine hepatocytes. Cytotoxicity evaluation of cultured cells (MTT assay, extracellular lactate dehydrogenase activity) was performed at 12, 24 and 48 h of exposure to alpha-AMA and/or antidotes. RESULTS: Following 24 and 48 h exposure there was a significant decline of hepatocyte viability and an increase of lactate dehydrogenase activity in groups exposed to alpha-AMA and in groups exposed simultaneously to alpha-AMA and antidotes. Moreover, hepatocyte viability and lactate dehydrogenase activity in all these groups were similar. Administration of studied antidotes without alpha-AMA, was not associated with any adverse effects in hepatocytes. CONCLUSION: All antidotes tested in this study against alpha-AMA were not effective in canine hepatocyte cultures.