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Plasma levels of a aspartate aminotransferase (AST), b alanine aminotransferase (ALT), c urea d creatinine e ratio AST/ALT and f total bilirubin in control, 3 × 2.5 mg/kg polymyxin B (Pol), 0.33 mg/kg α-amanitin (Ama), and α-amanitin plus polymyxin B (Ama + Pol) groups. Results are presented as mean ± standard deviation and were obtained from 4 to 5 animals from each treatment. Statistical comparisons were made using Kruskal–Wallis ANOVA on ranks followed by the Dunn’s post hoc test (*p < 0.05, Ama vs. control; #p < 0.05, Ama vs. Ama + Pol)
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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 g...
Citations
... Amanita phalloides poisoning is a serious health problem, accounting for over 90% of fatalities due to mushrooms [1][2][3]. Most cases of Amanita phalloides poisonings occur during the fruiting season, as these mushrooms may be easily mistaken for edible species [4]. ...
... The inhibition of RNA polymerase II is considered to be the main mechanism of toxicity, leading to the inhibition of protein synthesis and subsequent liver toxicity [7]. Other mechanisms, such as the formation of reactive oxygen species, caspase-3dependent apoptosis, and the upregulation of tumor necrosis factor-α, may be involved as well [1,5,6,9,10]. In patients, Amanita phalloides poisonings start with a latency phase followed by a gastrointestinal phase 12-24 h after the ingestion of the mushrooms [4,11]. ...
Introduction: Amanita phalloides poisoning is a serious health problem with a mortality rate of 10–40%. Poisonings are characterized by severe liver and kidney toxicity. The effect of Amanita phalloides poisonings on hematological parameters has not been systematically evaluated thus far. Methods: Patients with suspected Amanita phalloides poisonings were retrospectively selected from the hospital database of the University Medical Center Groningen (UMCG). Medical data—including demographics; liver, kidney, and blood parameters; treatment; and outcomes—were collected. The severity of the poisoning was scored using the poison severity score. Results: Twenty-eight patients were identified who were admitted to the UMCG with suspected Amanita phalloides poisoning between 1994 and 2022. A time-dependent decrease was observed for hemoglobin and hematocrit concentrations, leukocytes, and platelets. Six out of twenty-eight patients developed acute liver failure (ALF). Patients with ALF showed a higher increase in liver enzymes, international normalized ratios, and PSS compared to patients without ALF. Conversely, hemoglobin and platelet numbers were decreased even further in these patients. Three out of six patients with ALF died and one patient received a liver transplant. Conclusion: Our study shows that Amanita phalloides poisonings may be associated with hematotoxicity in patients. The quantification of hematological parameters is of relevance in intoxicated patients, especially in those with ALF.
... α-Amanitin is mainly responsible for the severe liver and kidney injury observed [6]. It is well established that α-Amanitin inhibits RNA polymerase II, thereby interfering with the transcription process [7]. RNA polymerase II transcribes all protein-coding genes and many noncoding RNAs in the eukaryotic genome. ...
... As a result, a series of proteins and protein complexes are required to interact with Pol II to regulate its activity and perform these essential functions [8]. However, research shows that even avoiding RNA polymerase II inactivation with structural inhibitors of α-Amatoxin did not alleviate late mortality in surviving animals [7,9,10], suggesting that occupancy inhibition of RNA polymerase II was not the only pathway of liver injury attributed to α-Amatoxin. ...
... The current study confirmed that α-Amatoxin causes damage by potentially inducing an acute inflammatory response [7], which is due to continuous release of such things as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin IL23A (IL-23A). JAK2 is a member of the Janus family of tyrosine kinases, the JAK2/STAT3 signaling pathway is a signal transduction pathway stimulated by cytokines, which was discovered in recent years, and it participates in many important organisms such as Inflammatory diseases, apoptosis and immune regulation. ...
... The hepatotoxicity of AMA primarily arises from its inhibition of RNA polymerase II activity, disrupting normal RNA synthesis in liver cells, ultimately leading to hepatocyte death [13]. However, the inhibition of RNA polymerase II is not the sole pathway through which α-amanitin-induced liver damage occurs [14][15][16]. There may be other therapeutic targets for α-amanitin-induced liver injury. ...
α-Amanitin is a representative toxin found in the Amanita genus of mushrooms, and the consumption of mushrooms containing α-Amanitin can lead to severe liver damage. In this study, we conduct toxicological experiments to validate the protective effects of Ganoderic acid A against α-amanitin-induced liver damage. By establishing animal models with different durations of Ganoderic acid A treatment and conducting a metabolomic analysis of the serum samples, we further confirmed the differences in serum metabolites between the AMA+GA and AMA groups. The analysis of differential serum metabolites after the Ganoderic acid A intervention suggests that Ganoderic acid A may intervene in α-amanitin-induced liver damage by participating in the regulation of retinol metabolism, tyrosine and tryptophan biosynthesis, fatty acid biosynthesis, sphingosine biosynthesis, spermidine and spermine biosynthesis, and branched-chain amino acid metabolism. This provides initial insights into the protective intervention mechanisms of GA against α-amanitin-induced liver damage and offers new avenues for the development of therapeutic drugs for α-Amanitin poisoning.
... Among all poisonous mushrooms, death caps (Amanita phalloides) are responsible for more than 90% of death 4 . Amatoxin poisoning is commonly associated with poor outcomes, mainly owing to the irreparable acute failure of the liver or kidney 5,6 . ...
... The toxic effects of AMA on humans are considered to be associated with the inhibition of RNA polymerase II (RNAP II) 7 , leading to the production of tumor necrosis factor-α (TNFα) 8 , oxidative stress 9 , and apoptosis 10 . Traditional therapies are often limited to the nonspecific decontamination of toxins along with symptomatic and supportive care 5 . During the past decades, several clinical drugs including silybin and penicillin have shown potent therapeutic efficacy on human amatoxin poisoning 11 , although the exact mechanisms of action remain unclear 12 . ...
... During the past decades, several clinical drugs including silybin and penicillin have shown potent therapeutic efficacy on human amatoxin poisoning 11 , although the exact mechanisms of action remain unclear 12 . Moreover, polymyxin B, identified as a potential RNAP II inhibitor in a virtual docking, has been shown to block AMA toxicity in mice 5 . However, specific antidotes targeting specific proteins that play critical roles in AMA toxicity are unavailable since a complete molecular understanding of AMA cytotoxicity is lacking. ...
The “death cap”, Amanita phalloides, is the world’s most poisonous mushroom, responsible for 90% of mushroom-related fatalities. The most fatal component of the death cap is α-amanitin. Despite its lethal effect, the exact mechanisms of how α-amanitin poisons humans remain unclear, leading to no specific antidote available for treatment. Here we show that STT3B is required for α-amanitin toxicity and its inhibitor, indocyanine green (ICG), can be used as a specific antidote. By combining a genome-wide CRISPR screen with an in silico drug screening and in vivo functional validation, we discover that N-glycan biosynthesis pathway and its key component, STT3B, play a crucial role in α-amanitin toxicity and that ICG is a STT3B inhibitor. Furthermore, we demonstrate that ICG is effective in blocking the toxic effect of α-amanitin in cells, liver organoids, and male mice, resulting in an overall increase in animal survival. Together, by combining a genome-wide CRISPR screen for α-amanitin toxicity with an in silico drug screen and functional validation in vivo, our study highlights ICG as a STT3B inhibitor against the mushroom toxin.
... 3,6,7 Three groups of toxins have been identified as being responsible for their toxicity: amatoxins, phallotoxins and virotoxins. 8 Among them, amatoxins are the only toxins found in common among the three genera and would be responsible for organ damage. 3 The main molecular mechanism involved in amatoxin-induced toxicity is their irreversible binding to DNA-dependent RNA polymerase II in eukaryotic cells, causing a progressive decrease in mRNA, leading to a decrease in protein synthesis and cell death. ...
... Finally, late presentations (maximum at 36 h) are also found in our series but also in other published case series. 8,18 In 78% of cases, patients presented with hepatitis and live injury. It was observed, on average, after 60 h, with a nearly synchronous increase in AST and ALT. ...
Cyclopeptide mushroom poisoning is responsible for 90‐95% of deaths from macrofungi ingestion. The main objectives of this study are to describe cases of cyclopeptide mushroom poisoning and to determine risk factors that may influence the severity/mortality of poisoned patients. We included all cases of amatoxin toxicity reported to two french Poison Centers from 2013 through 2019. We compared the severity with the Poison Severity Score (PSS) and the outcomes of patients using simple logistic regression and multinomial logistic regression. We included 204 cases of amatoxin toxicity. More than three‐quarters developed an increase in AST and/or ALT (78.1%) and over half developed a decrease in prothrombin ratio (<70%: 53%) and/or Factor V (<70%: 54%). One third developed an acute renal injury (AKI). Twelve patients (5.9%) developed post‐poisoning sequelae (persistent kidney injury more than one month after ingestion and liver transplant). Five patients (2.5%) received a liver transplant and 9 died (4.4%).The mean time to onset of digestive disorders was shorter in PSS2 and PSS3‐4 patients (10.9±3.9/11.3±6.3 hours) than in PSS1 patients (14±6.5 hours; p<0.05). Patients who died or developed post‐poisoning sequelae had more frequently cardiovascular comorbidities compared with recovered patients (60.0% versus 29.5%; p<0.01).
... The non-toxic cyclic decapeptide antamanide and the highly toxic bicyclic heptapeptide phalloidin from the poisonous mushroom Amanita phalloides use overlapping, and possibly identical, uptake systems as bile acids to enter hepatocytes [319][320][321][322][323][324] . ...
Reporter-kodierende Hepatitis-B-Virus-(HBV)-Varianten sollten nach Infektion einen sensitiven und quantitativen Nachweis der Reporterexpression ausgehend von der kovalent geschlossenen, zirkulären (covalently closed circular, ccc)DNA, dem persistierenden viralen Minichromosom, ermöglichen. Frühere Versuche zur Entwicklung von Reporter-(r)HBV-Varianten waren aufgrund des kleinen und komplex aufgebauten Genoms wenig erfolgreich. Das Einbringen transgener Sequenzen führt in der Regel zu multiplen Defekten, die eine Transkomplementierung mit Vollgenom-HBV-Helferplasmiden verlangen.
In dieser Arbeit wurden verschiedene Reportervektoren entwickelt, bei denen das HBc-Gen größteneils durch ein Transgen für Gaussia-Dura-Luciferase (Luc) ersetzt wurde. Die Replikation sollte ausschließlich von in trans bereitgestelltem HBc abhängen. Die Translation sowohl des Luc-Reporters wie auch von Pol wurde durch unterschiedliche Kombinationen von Translations-Kontrollelementen ermöglicht, d.h. 2A-Peptide, kleine interne ribosomale Eintrittsstellen (small internal ribosome entry sites, sIRESs) oder "Slip"-Sequenzen zur Induktion einer programmierten ribosomalen Leserasterverschiebung (programmed ribosomal frameshifting, PRF). Alle Vektoren wurden auf Reporterexpression, Replikation und Bildung der funktionellen Genomkonformation, der relaxierten zirkulären (relaxed circular, rc)DNA, untersucht. Ein Reporterkonstrukt, in dem Luc in-frame mit N-terminalen HBc-Resten kotranslatiert und die Pol-Transation über ein Slip-Motiv gesteuert wird, wurde weiter optimiert. Dies beinhaltete die Verkürzung der verbliebenen HBc-Restsequenz sowie die Deletion nicht-essentieller Luc-interner Sequenzen.
Infektiosität und infektionsabhängige Reporterexpression wurden durch Inokulation von HepG2-NTCP-Zellen mit rHBV untersucht. Eindeutige Ergebnisse wurden zunächst durch in den rHBV-Inokula enthaltenes, während der Virusproduktion von rHBV-Plasmid transfizierten Zellen exprimiertes Luc Protein erschwert. Aus mehreren Methoden zur Entfernung oder Inaktivierung dieses Luc Proteins erwies sich ein GluC-Protease-Verdau der Inokula als am effizientesten, wobei die rHBV-Infektiosität vollständig erhalten blieb.
Ansätze zur Steigerung der Virustiter beinhalteten die rHBV-Produktion durch Transduktion von
Hepatomzellen mit rekombinanten Adeno-assoziiertes Virus-(rAAV)-rHBV-Partikeln sowie die
lentivirale Integration einer rHBV-kodierenden TetON-Expressionskassette in das Genom von HepG2-HBc-Zellen, die konstitutiv HBc exprimieren. Schlussendlich wurden die besten Ergebnisse jedoch mittels konventioneller Kotransfektion unter Anwendung eines optimierten Protokolls erzielt.
Andere Quellen als cccDNA für die Luc-Expression wurden experimentell durch Mutationsanalysen
ausgeschlossen, einschließlich durch rHBV-Mutanten mit Defekten in der Bildung von rcDNA, dem
authentischen cccDNA-Vorläufer.
Die Eignung von rHBV als wt HBV-Surrogat wurde durch den Nachweis der antiviralen Wirkung zweier Kapsid-Assemblierungs-Modulatoren (capsid assembly modulators, CAMs) demonstriert, wobei die Lumineszenz vergleichbare Ergebnisse wie die Expression des HBV-Oberflächenantigens (surface, HBs) und des e-Antigens (HBe) nach einer wt HBV-Infektion zeigte. Darüber hinaus produzierte die Transkomplementation von rHBV mit HBc-Varianten mit Mutationen der hydrophoben Tasche, L60G und L60W, nackte Kapside, aber keine umhüllte Virionen, analog zu früheren Ergebnissen mit wt HBV. Eine Inokulation mit rHBV L60G oder rHBV L60W erzeugte dementsprechend keine Lumineszenz.
Mit rHBV als Werkzeug zur Identifizierung neuer antiviraler Substanzen wurde die Wirkung des
zyklischen Decapeptids Antamanid aus dem Pilz Amanita phalloides auf die HepG2-NTCP-Infektion
analysiert, wobei mikromolare Konzentrationen dosisabhängig die Reporteraktivität verringerten.
Schließlich wurden in einem knockdown-Ansatz zelluläre Wirtsfaktoren, darunter bereits beschriebene HBV-Interaktionspartner wie auch neue Kandidaten, durch lentivirale Expression von shRNAs in HepG2-NTCP Zellen gezielt herabreguliert, um ihren Einfluss auf die Bildung von cccDNA zu untersuchen. Die Inokulation dieser Zellpools identifizierte u.a. die Flap-Endonuklease 1 (FEN1) als cccDNA-relevanten Wirtsfaktur, in Einklang mit früheren Studien. Somit ist rHBV auch zur Charakterisierung von HBV-Wirt-Interaktionen geeignet.
Zusammengefasst wurde in dieser Arbeit ein neues HBV-Reportersystem entwickelt, welches eine
sensitive und quantitative Detektion der cccDNA-Bildung nach Infektion ermöglicht. Validierung und
Optimierung erfolgten im HepG2-NTCP-Zellkultursystem, dem derzeit primären Infektionsmodell.
Dabei reproduzierte rHBV diverse, zuvor für wt HBV beschriebene Ergebnisse und demonstrierte damit sein Potenzial als wt HBV Surrogat. Schließlich erlaubte rHBV die Ermittlung der unbekannten antiviralen Kapazität von Antamanid. Künftig sollte dieses neue rHBV-System dazu beitragen, die komplexen Wechselwirkungen zwischen HBV und Wirt aufzuklären und ein besseres Verständnis der cccDNA-Bildung zu ermöglichen sowie die Entwicklung neuer therapeutischer Ansätze zu fördern.
... Moreover, polymyxin B significantly reduced α-amanitin-induced injury in the liver and kidney, as shown by histological and hepatic transaminase plasma data. In survival assays, all animals exposed to α-amanitin died within 5 days, whereas 50% survived for 30 days when polymyxin B was given 4, 8, and 12 h after α-amanitin (Garcia et al. 2015). In 2019, the research group conducted in-depth research on the antidote of α-amanitin. ...
... Juliana Garcia et al. used the combination of polymyxin B and methylprednisone to treat poisoning caused by α-amanitin (Garcia et al. 2019). Polymyxin B was proved to be able to competitively bind RNA polymeraseII sites with α-amanitin, thus playing a therapeutic role (Garcia et al. 2015). Methylprednisolone was proved to be a potent suppressive agent of NTCP in an in vitro experiment, which prevented α-amanitin from entering cells, thus achieving the therapeutic effect (Dong et al. 2013). ...
Amanita poisoning has a high mortality rate. The α-amanitin toxin in Amanita is the main lethal toxin. There is no specific detoxification drug for α-amanitin, and the clinical treatment mainly focuses on symptomatic and supportive therapy. The pathogenesis of α-amanitin mainly includes: α-amanitin can inhibit the activity of RNA polymeraseII in the nucleus, including the inhibition of the largest subunit of RNA polymeraseII, RNApb1, bridge helix, and trigger loop. In addition, α-amanitin acts in vivo through the enterohepatic circulation and transport system. α-Amanitin can cause the cell death. The existing mechanisms of cell damage mainly focus on apoptosis, oxidative stress, and autophagy. In addition to the pathogenic mechanism, α-amanitin also has a role in cancer treatment, which is the focus of current research. The mechanism of action of α-amanitin on the body is still being explored.
... The immunohistochemistry analysis was performed, as described by others [16], to quantify M1 and M2 macrophage subtypes and neutrophils present in the damaged-muscle area. Briefly, slides were deparaffinized and put in a pressure cooker for 20 min in 10 mM citrate buffer, pH 6.0, for the antigen-retrieval procedure. ...
This study investigated whether sedentary behaviour modulates skeletal-muscle repair and tissue inflammatory response after cardiotoxin (CTX)-induced injury. Singly caged rats spent 8 weeks either as a sedentary group (SED, n = 15) or as a control group (EX, n = 15)—caged with running wheels for voluntary running. All rats had each tibial anterior muscle infused either with CTX (CTX; right muscle) or saline solution (Sham; left muscle) and were sacrificed (n = 5 per group) on the 1st, 7th, and 15th day post-injection (dpi). Histological and immunohistochemical analyses were used to calculate myotube percentage and fibrosis accretion, and quantify the number of neutrophils and M1 and M2 macrophage subtypes. The SED group showed an increased number of both neutrophils and M1 macrophages (7th and 15th dpi) compared to the EX group (p < 0.01). The EX group showed an increased number of M2 macrophages on the 1st dpi. On the 7th dpi, the SED group showed a lower myotube percentage compared to the EX group (p < 0.01) and on the 15th dpi showed only 54% of normal undamaged fibres compared to 90% from the EX group (p < 0.01). The SED group showed increased fibrosis on both the 7th and 15th dpi. Our results show that sedentary behaviour affects the inflammatory response, enhancing and prolonging the Th1 phase, and delays and impairs the SMR process.
... Considering that decreased mRNA synthesis is the event directly downstream of the primary interaction between α-amanitin and its target molecule, RNAP II, changes in transcriptional levels are of particular interest (Larson 2011;Garcia et al. 2015b). To some extent, mRNA change has potential as an indicator, reflecting and describing the liver toxicity of α-amanitin at a molecular level. ...
... To some extent, mRNA change has potential as an indicator, reflecting and describing the liver toxicity of α-amanitin at a molecular level. However, statistically significant decreases in the expression of individual mRNAs encoding stably expressed proteins in the cell (such as GAPDH or β-actin) or that of total RNA in tissues after α-amanitin exposure have been challenging to observe (Garcia et al. 2015b). Transcriptome sequencing, as an omics method that focuses on mRNA expression levels (sequencing after isolation and purification of mRNA from total RNA), allows the identification of genes with differential expression (i.e., DEGs) before and after exposure. ...
... b Enrichment map of pathways from the above subcluster, mainly including the mTOR, MAPK, AMPK, and insulin signaling pathways (color figure online) of decreased mRNA and decreased total protein in liver. Consistent with our findings, a similar significant liver/body weight ratio decrease in an early period after α-amanitin exposure in mice was observed in previous studies (Garcia et al. 2015b). (4) Liver cell vacuolization was observed in pathology at 24 h. ...
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.
... This blockage in RNAP II transcription leads to apoptosis (Arima et al., 2005;Leu and George, 2007). Inflammation and oxidative stress also contribute to α-amanitin toxicity as our reports and others have shown (Garcia et al., 2015c(Garcia et al., , 2019Leist et al., 1994Leist et al., , 1997Zheleva et al., 2007), dependent or not on previous RNAP II inhibition. ...
... Our past approach regarding the discovery of new antidotes took the notion that the ideal therapeutic approach against A. phalloides intoxications would be to displace and/or compete with α-amanitin binding to RNAP II without impairing its normal transcription activity. Conversely, both in silico and in vivo, polymyxin B was revealed to be a promising antidote against a lethal dose of amanitin (Garcia et al., 2015c(Garcia et al., , 2019 although it did not counteract the toxin's toxicity nor prevented RNAP II inhibition in vitro at relevant clinical concentrations (Rodrigues et al., 2020). Thus, we hypothesised another possible explanation to the therapeutic relevant results found: the inhibition of transporting systems mediating the uptake of α-amatoxin into hepatic and kidney cells. ...
... Notwithstanding the apparent discordance between the in vivo, in silico and in vitro results, our innovative approach based on the screening of clinical drugs that show bioisosterism with amatoxins, namely reporting to its binding with RNAP II, revealed an antidote that counteracted amanitin's lethality (Garcia et al., 2015c(Garcia et al., , 2019. Therefore, we continued to pursue the same strategies, evaluating the bioisosterism of other drugs in in silico models, focusing on drugs already in clinical use and already tested for safety, to assess a putative competition and displacement from amatoxins binding site with RNAP II. ...
Amanita phalloides is one of the most toxic mushrooms worldwide, being responsible for the majority of human fatal cases of mushroom intoxications. α-Amanitin, the most deleterious toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and renal failure. Herein, we used cyclosporine A that showed potential to displace RNAP II α-amanitin in silico. That potential was not confirmed either by the incorporation of ethynyl-UTP or by the monitoring of fluorescent RNAP II levels. Nevertheless, concomitant incubation of cyclosporine A with α-amanitin, for a short period, provided significant protection against its toxicity in differentiated HepaRG cells. In mice, the concomitant administration of α-amanitin [0.45 mg/kg intraperitoneal (i.p.)] with cyclosporine A (10 mg/kg i.p. plus 2 × 10 mg/kg CsA i.p. at 8 and 12 hours post α-amanitin) resulted in the full survival of α-amanitin-intoxicated mice, even 30 days after the toxin's administration. Since α-amanitin is a substrate of the organic-anion-transporting polypeptide 1B3 and cyclosporine A its inhibitor and a potent anti-inflammatory agent, we hypothesize that these mechanisms are responsible for the protection observed. These results indicate a potential antidotal effect of cyclosporine A and its safety profile advocate for its use at an early stage of α-amanitin intoxications.
