Search Results (33)

Amanita phalloides is well-established as one of the most poisonous mushrooms; toxicity from ingestion was reported as early as the first century. Although native to Europe, this ectomycorrhizal fungus has been widely spread and is responsible for liver toxicity in many parts of the world. Toxicity is characterized by delayed gastrointestinal symptoms mimicking acute gastroenteritis followed by severe hepatotoxicity and liver failure with consequent multi-organ failure. The primary mechanism of liver toxicity is considered to be the inhibition of RNA polymerase II with consequent hepatocyte apoptosis. Treatment measures include supportive measures such as rehydration and correction of electrolytes on initial presentation, activated charcoal and lavage to decrease absorption, extracorporeal purification methods such as plasmapheresis, fractionated plasma separation and adsorption, and molecular adsorbent recirculating system, as well as drug therapies including antibiotics, N-acetylcysteine, and silibinin. Liver transplantation is required in those with acute liver failure and poor prognostic features. Here, we reviewed the basic biology, pathophysiology, and molecular mechanisms of Amanita phalloides liver toxicity, as well as available treatments. Full article

A comprehensive analytical method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous detection of 12 mushroom toxins (ibotenic acid, muscimol, muscarine, β-amanitin, α-amanitin, desoxoviroidin, γ-amanitin, phallisacin, illudin S, phallacidin, phalloidin and illudin M) in mushrooms, serum, urine and simulated gastric fluid. The samples were extracted with water or acetonitrile solution, and the serum sample was further purified with PSA sorbent. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column with gradient elution using methanol and water containing 1 mM ammonia fluoride as a mobile phase. Mass spectrometric acquisition was performed in electrospray positive ionization mode. Good linearities (R² > 0.994) were obtained for 12 toxins over the range of 0.05~200 µg/L. Matrix-matched calibration curves were used for quantification. The method limits of quantification were 0.01~0.2 mg/kg for mushrooms and 0.15~2.0 µg/L for three biological liquid samples. The mean recoveries of 12 target toxins (spiked at three concentration levels) ranged from 73.0% to 110.3%, with relative standard deviations not exceeding 19.4%, which meets the requirements for the determination of trace compounds in a biological matrix. This method was applied to the analysis of mushroom samples from Yunnan Province. As a result, 11 toxins, not including illudin M, were detected with a concentration range of 0.61~2143 mg/kg. Full article

Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and β-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or β-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. β-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism. Full article

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. Full article

α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin’s activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT–formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 μg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors. Full article

Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and certain groups of animals) stand out. Amatoxin-containing mushrooms and the poisonings caused by them stand out from the higher fungi, the mushrooms. There are already historical data and records about such poisonings, but scientific research on the responsible molecules began in the middle of the last century. The goals of this review work are as follows: presentation of the cosmopolitan mushroom species that produce amanitins (which are known from certain genera of four mushroom families), an overview of the chemical structure and specific properties of amanitins, a summary of the analytical methods applicable to them, a presentation of the “medical history” of poisonings, and a summary of the therapeutic methods used so far. The main responsible molecules (the amanitins) are bicyclic octapeptides, whose structure is characterized by an outer loop and an inner loop (bridge). It follows from the unusual properties of amanitins, especially their extreme stability (against heat, the acidic pH of the medium, and their resistance to human, and animal, digestive enzymes), that they are absorbed almost without hindrance and quickly transported to our vital organs. Adding to the problems is that accidental consumption causes no noticeable symptoms for a few hours (or even 24–36 h) after consumption, but the toxins already damage the metabolism of the target organs and the synthesis of nucleic acid and proteins. The biochemical catastrophe of the cells causes irreversible structural changes, which lead to necrotic damage (in the liver and kidneys) and death. The scientific topicality of the review is due to the recent publication of new data on the probable antidote molecule (ICR: indocyanine green) against amanitins. Further research can provide a new foundation for the therapeutic treatment of poisonings, and the toxicological situation, which currently still poses a deadly threat, could even be tamed into a controllable problem. We also draw attention to the review conclusions, as well as the mycological and social tasks related to amanitin poisonings (prevention of poisonings). Full article

A 10-year-old, female spayed Labrador Retriever was referred for acute hepatopathy and urinary retention. Blood work from the initial presentation (day 0) revealed a severe, mixed hepatopathy. Over the course of the patient’s hospitalization, the patient developed liver insufficiency. Urine was submitted for toxicological screening and revealed detection of a trace concentration of alpha-amanitin. The patient was treated supportively for alpha-amanitin intoxication and was discharged from the hospital on day 8, with most biochemical parameters being markedly improved. The patient was persistently hyporexic at the time of discharge. On day 15, at a recheck appointment, the patient had lost 2.4 kg and liver enzymology revealed improved values. On day 24, the patient was presented for anorexia and vomiting and had lost another 2.3 kg. Blood work and endocrinological testing at that time were consistent with hypoadrenocorticism. The patient was started on glucocorticoids and mineralocorticoids. At day 106, the patient was doing well clinically while receiving monthly mineralocorticoids and daily glucocorticoids. This case report is the first to describe the chronological association between alpha-amanitin-induced liver dysfunction and the subsequent development of adrenal insufficiency in a dog. Full article

Targeting fibroblast growth factor receptor 1 (FGFR1) is a promising therapeutic strategy for various cancers associated with alterations in the FGFR1 gene. In this study, we developed a highly cytotoxic bioconjugate based on fibroblast growth factor 2 (FGF2), which is a natural ligand of this receptor, and two potent cytotoxic drugs—α-amanitin and monomethyl auristatin E—with completely independent mechanistic modes of action. Utilizing recombinant DNA technology, we produced an FGF2 N- to C-end dimer that exhibited superior internalization capacity in FGFR1-positive cells. The drugs were site-specifically attached to the targeting protein using SnoopLigase- and evolved sortase A-mediated ligations. The resulting dimeric dual-warhead conjugate selectively binds to the FGFR1 and utilizes receptor-mediated endocytosis to enter the cells. Moreover, our results demonstrate that the developed conjugate exhibits about 10-fold higher cytotoxic potency against FGFR1-positive cell lines than an equimolar mixture of single-warhead conjugates. The diversified mode of action of the dual-warhead conjugate may help to overcome the potential acquired resistance of FGFR1-overproducing cancer cells to single cytotoxic drugs. Full article

Ganoderma lucidum (G. lucidum) has been widely used for its health benefits as an edible and traditional medicinal mushroom for thousands of years in Asian countries. It is currently used as a nutraceutical and functional food owing to its major bioactive compounds, polysaccharides and triterpenoids. G. lucidum exhibits a broad range of hepatoprotective impacts in various liver disorders, such as hepatic cancer, nonalcoholic fatty liver disease (NAFLD), alcohol-induced liver disease, hepatitis B, hepatic fibrosis, and liver injury induced by carbon tetrachloride (CCl4) and α-amanitin. G. lucidum protects the liver through a broad range of mechanisms that include the modulation of liver Phase I and II enzymes, the suppression of β-glucuronidase, antifibrotic and antiviral actions, the regulation of the production of nitric oxide (NO), the maintenance of hepatocellular calcium homeostasis, immunomodulatory activity, and scavenging free radicals. G. lucidum could signify an encouraging approach for the management of various chronic hepatopathies, and its potential mechanisms make it a distinctive agent when used alone or with other drugs and applied as a functional food, nutraceutical supplement, or adjuvant to modern medicine. This review summarizes the hepatoprotective properties of G. lucidum with its various mechanisms of action on different liver ailments. Biologically active substances derived from G. lucidum are still being studied for their potential benefits in treating different liver ailments. Full article

The purpose of this study is to investigate the difference of in vitro–in vivo correlation of α-amanitin from clearance perspectives as well as to explore the possibility of extra-hepatic metabolism of α-amanitin. First, a liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) method for α-amanitin in rat plasma was developed and applied to evaluate the in vitro liver microsomal metabolic stability using rat and human liver microsomes and the pharmacokinetics of α-amanitin in rat. The predicted hepatic clearance of α-amanitin in rat liver microsomes was quite low (5.05 mL/min/kg), whereas its in vivo clearance in rat (14.0 mL/min/kg) was close to the borderline between low and moderate clearance. To find out the difference between in vitro and in vivo metabolism, in vitro and in vivo metabolite identification was also conducted. No significant metabolites were identified from the in vivo rat plasma and the major circulating entity in rat plasma was α-amanitin itself. No reactive metabolites such as GSH-adducts were detected either. A glucuronide metabolite was newly identified from the in vitro liver microsomes samples with a trace level. A semi-mass balance study was also conducted to understand the in vivo elimination pathway of α-amanitin and it showed that most α-amanitin was mainly eliminated in urine as intact which implies some unknown transporters in kidney might play a role in the elimination of α-amanitin in rat in vivo. Further studies with transporters in the kidney would be warranted to figure out the in vivo clearance mechanism of α-amanitin. Full article

Mushroom poisoning remains a serious food safety and health concern in some parts of the world due to its morbidity and mortality. Identification of mushroom toxins at an early stage of suspected intoxication is crucial for a rapid therapeutic decision. In this study, a new extraction method was developed to determine α- and β-amanitin in mushroom samples collected from central Portugal. High-performance liquid chromatography with in-line ultraviolet and electrochemical detection was implemented to improve the specificity of the method. The method was fully validated for linearity (0.5–20.0 µg·mL⁻¹), sensitivity, recovery, and precision based on a matrix-matched calibration method. The limit of detection was 55 µg mL⁻¹ (UV) and 62 µg mL⁻¹ (EC) for α-amanitin and 64 µg mL⁻¹ (UV) and 24 µg mL⁻¹ (EC) for β–amanitin. Intra- and inter-day precision differences were less than 13%, and the recovery ratios ranged from 89% to 117%. The developed method was successfully applied to fourteen Amanita species (A. sp.) and compared with five edible mushroom samples after extraction with Oasis^® PRIME HLB cartridges without the conditioning and equilibration step. The results revealed that the A. phalloides mushrooms present the highest content of α- and β-amanitin, which is in line with the HPLC-DAD-MS. In sum, the developed analytical method could benefit food safety assessment and contribute to food-health security, as it is rapid, simple, sensitive, accurate, and selectively detects α- and β-amanitin in any mushroom samples. Full article

The Spheroid Reservoir Bioartificial Liver (SRBAL) is an innovative treatment option for acute liver failure (ALF). This extracorporeal support device, which provides detoxification and other liver functions using high-density culture of porcine hepatocyte spheroids, has been reported in three randomized large animal studies. A meta-analysis of these three preclinical studies was performed to establish efficacy of SRBAL treatment in terms of survival benefit and neuroprotective effect. The studies included two hepatotoxic drug models of ALF (D-galactosamine, α-amanitin/lipopolysaccharide) or a liver resection model (85% hepatectomy) in pigs or monkeys. The SRBAL treatment was started in three different settings starting at 12 h, 24 h or 48 h after induction of ALF; comparisons were made with two similar control groups in each model. SRBAL therapy was associated with significant survival and neuroprotective benefits in all three animal models of ALF. The benefits of therapy were dose dependent with the most effective configuration of SRBAL being continuous treatment of 24 h duration and dose of 200 g of porcine hepatic spheroids. Future clinical testing of SRBAL in patients with ALF appears warranted. Full article

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells. Full article

The toxicokinetics of β-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. β-amanitin disappeared rapidly from plasma with a half-life of 18.3–33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3–9.4%, which resulted in 72.4% of fecal recovery from orally administered β-amanitin. Tissue-to-plasma AUC ratios of orally administered β-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, β-amanitin weakly or negligibly inhibited major cytochrome P450 and 5′-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by β-amanitin exposure. Full article

Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation. Full article