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OBJECTIVE: Amatoxin-containing mushroom poisoning is a significant threat to public health worldwide. We report a mass poisoning of Galerina sulciceps-like mushrooms (Galerina cf. sulciceps) in Luzhou, Sichuan Province, China, aiming to offer insights for future prevention and treatment strategies. METHODS: We performed a retrospective survey of mass mushroom poisoning patients admitted to our hospital. The demographic data, clinical presentations, laboratory findings, therapeutic measures and prognostic information were collected and analyzed. We used the 2020 Chinese consensus on the clinical diagnosis and treatment of amatoxin-containing mushroom poisoning to assess the severity of poisoning. Mushrooms were examined through morphological analysis, molecular biology identification, and toxin detection. RESULTS: Our patient cohort consisted of nine males and six females, with mean (±SD) age of 34.9 ± 13.0 years. Gastrointestinal symptoms were the first to manifest, with mean (±SD) latency period of 13.4 ± 3.9 h. The majority of patients (86.7%) experienced nausea, vomiting, and diarrhea. Liver dysfunction was noted in 66.7% of patients, and thrombocytopenia was present in 26.7% of patients. In terms of the severity of poisoning, there were 10 mild cases and 5 severe cases. The mushrooms were provisionally labeled as Galerina cf. sulciceps, containing the toxins α-amanitin, ß-amanitin, and γ-amanitin. All patients eventually recovered. DISCUSSION: We report what appears to be a new type of mushroom that is morphologically and phylogenetically similar to the known Galerina sulciceps, but further study is required to determine if it represents a distinct species. CONCLUSION: This poisoning event was caused by unintentional ingestion of Galerina cf. sulciceps, an amatoxin-containing mushroom. Early symptoms are primarily gastrointestinal, with acute liver damage and coagulopathy being the main toxic effects. Thrombocytopenia is also prominent, particularly in severe cases. Accurate assessment and prompt, individualized, and intensive treatment are crucial for managing patients with acute Galerina cf. sulciceps poisoning effectively.

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BACKGROUND: Mushroom poisoning poses a significant global health concern, with high morbidity and mortality rates. The primary lethal toxins responsible for this condition are alpha-amanitin (ɑ-AMA) and beta-amanitin (ß-AMA). As a promising bio-recognition molecules in biosensors, aptamers, have been broadly used in the field of food detection. However, the current SELEX-based methods for screening aptamers for structurally similar small molecules were limited by the labelling or salt ion induction. In this study, we aimed to develop a novel label-free SELEX strategy for the screening of aptamers with high affinity and constructed new aptasensors for the detection of ɑ-AMA and ß-AMA. RESULTS: A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for ɑ-AMA and ß-AMA. Only 18 rounds of SELEX were required to obtain new aptamers. The candidate aptamers were analyzed by colloidal gold assay, and the sequences of ɑ-30 and ß-37 displayed great affinity with Kd values of 22.26 nM and 23.32 nM, respectively, without interference from botanical toxins. Notably, the truncated aptamers ɑ-30-2 (50 bp) and ß-37-2 (57 bp) exhibited higher affinity than their original counterpart (79 bp). Subsequently, the selected aptamers were utilized to construct recognition probes for electrochemical aptasensors based on hairpin cyclic cleavage of substrates by Cu2+ dependent DNAzyme and Exo I-triggered recycling cascades. The detection platform showed excellent analytical performance with limits of detection as low as 4.57 pg/mL (ɑ-AMA) and 8.49 pg/mL (ß-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples. SIGNIFICANCE: This work developed a simple and efficient label-free SELEX method for screening new aptamers for ɑ-AMA and ß-AMA, which employed the positively charged AuNPs as the screening medium, without the need for chemical labelling of libraries or induction of salt ions. Furthermore, two novel electrochemical aptasensors were developed based on our newly obtained aptamers, which offer the new biosensing tool for ultrasensitive detection of the AMA poisoning, showing great potential in practical applications.

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Drug conjugates are obtained from tumor-located vectors connected to cytotoxic agents via linkers, which are designed to deliver hyper-toxic payloads directly to targeted cancer cells. These drug conjugates include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), nucleic acid aptamer-drug conjugates (ApDCs), and virus-like drug conjugate (VDCs), which show great therapeutic value in the clinic. Drug conjugates consist of a targeting carrier, a linker, and a payload. Payloads are key therapy components. Cytotoxic molecules and their derivatives derived from natural products are commonly used in the payload portion of conjugates. The ideal payload should have sufficient toxicity, stability, coupling sites, and the ability to be released under specific conditions to kill tumor cells. Microtubule protein inhibitors, DNA damage agents, and RNA inhibitors are common cytotoxic molecules. Among these conjugates, cytotoxic molecules of natural origin are summarized based on their mechanism of action, conformational relationships, and the discovery of new derivatives. This paper also mentions some cytotoxic molecules that have the potential to be payloads. It also summarizes the latest technologies and novel conjugates developed in recent years to overcome the shortcomings of ADCs, PDCs, SMDCs, ApDCs, and VDCs. In addition, this paper summarizes the clinical trials conducted on conjugates of these cytotoxic molecules over the last five years. It provides a reference for designing and developing safer and more efficient conjugates.

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α-amanitin (AMA) is a hepatotoxic mushroom toxin responsible for over 90% of mushroom poisoning fatalities worldwide, seriously endangering human life and health. Few evidences have indicated that AMA leads to inflammatory responses and inflammatory infiltration in vitro and in vivo. However, the molecular mechanism remains unknown. In this study, human hepatocellular carcinomas cells (HepG2) were exposed to AMA at various concentrations for short period of times. Results revealed that AMA increased ROS production and elevated the releases of malondialdehyde (MDA) and lactate dehydrogenase (LDH), resulting in oxidative damage in HepG2 cells. Also, AMA exposure significantly increased the secreted levels of inflammatory cytokines and activated the NLRP3 inflammasome. The inflammatory responses were reversed by NLRP3 inhibitor MCC950 and NF-κB inhibitor Bay11-7082. Additionally, N-acetylcysteine (NAC) blocked the upregulation of the NF-κB/NLRP3 signaling pathway and remarkably alleviated the inflammatory response. These results demonstrated that AMA could induce inflammation through activating the NLRP3 inflammasome triggered by ROS/NF-κB signaling pathway. Our research provides new insights into the molecular mechanism of AMA-induced inflammation damage and may contribute to establish new prevention strategies for AMA hepatotoxicity.

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OBJECTIVE: The hepatoprotective effects of resveratrol against α-Amanitin (α-AMA)-induced liver toxicity were investigated in an experimental rat model, focusing on oxidative stress, inflammation, apoptosis, and liver function. METHODS: Thirty-two male Sprague-Dawley rats were divided into four groups (n = 8 per group): Control, resveratrol, α-AMA, and resveratrol+α-AMA. The resveratrol group received 20 mg/kg resveratrol orally for 7 days. The α-AMA group received 3 mg/kg α-AMA intraperitoneally on the 8th day. The resveratrol+α-AMA group received 20 mg/kg resveratrol orally (7 days) followed by 3 mg/kg α-AMA intraperitoneally on the 8th day. Liver tissues and blood samples were collected 48 h after α-amanitin administration for histopathological, immunohistochemical (NFkB, LC3B), and biochemical analyses (GSH, MDA, CAT, GPx, MPO, NOS, AST, ALT). RESULTS: α-AMA significantly increased AST and ALT levels, oxidative stress marker (MDA), and inflammatory marker (MPO), while reducing antioxidant levels (GSH, CAT, GPx) and NOS concentration (P < 0.001 for all parameters). Histopathological analysis showed severe liver damage with increased NFkB and LC3B expression. resveratrol treatment significantly reduced AST and ALT levels (P < 0.01 for both parameters), decreased MDA and MPO levels, and increased NOS concentration, GSH, CAT, and GPx levels (P < 0.05 for all parameters). Reduced NFkB and LC3B expression in the resveratrol+α-AMA group and showed histopathological improvements. CONCLUSION: Resveratrol demonstrated substantial hepatoprotective effects against α-AMA induced liver toxicity by reducing oxidative stress, inflammation, and apoptosis, and improving liver function. These findings suggest that resveratrol could be a potential therapeutic agent for treating liver damage caused by potent hepatotoxins like α-AMA.

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α-Amanitin and ß-amanitin, two of the most toxic amatoxin compounds, typically coexist in the majority of Amanita mushrooms. The aim of this study was to use a newly developed ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method to determine the toxicokinetics and tissue distribution of α- and ß-amanitin following single or combined oral (po) administration in mice. α-Amanitin and ß-amanitin administered at 2 or 10 mg/kg doses showed similar toxicokinetic profiles, except for peak concentration (Cmax). The elimination half-life (t1/2) values of α-amanitin and ß-amanitin in mice were 2.4-2.8 h and 2.5-2.7 h, respectively. Both α- and ß-amanitin were rapidly absorbed into the body, with times to reach peak concentration (Tmax) between 1.0 and 1.5 h. Following single oral administration at 10 mg/kg, the Cmax was significantly lower for α-amanitin (91.1 µg/L) than for ß-amanitin (143.1 µg/L) (p < 0.05). The toxicokinetic parameters of α-amanitin, such as t1/2, mean residence time (MRT), and volume of distribution (Vz/F) and of ß-amanitin, such as Vz/F, were significantly different (p < 0.05) when combined administration was compared to single administration. Tissues collected at 24 h after po administration revealed decreasing tissue distributions for α- and ß-amanitin of intestine > stomach > kidney > lung > spleen > liver > heart. The substantial distribution of toxins in the kidney corresponds to the known target organs of amatoxin poisoning. The content in the stomach, liver, and kidney was significantly higher for of ß-amanitin than for α-amanitin at 24 h following oral administration of a 10 mg/kg dose. No significant difference was detected in the tissue distribution of either amatoxin following single or combined administration. After po administration, both amatoxins were primarily excreted through the feces. Our data suggest the possibility of differences in the toxicokinetics in patients poisoned by mushrooms containing both α- and ß-amanitin than containing a single amatoxin. Continuous monitoring of toxin concentrations in patients' blood and urine samples is necessary in clinical practice.

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The consumption of mushrooms containing α-Amanitin (α-A) can lead to severe liver damage. In this study, toxicological experiments were conducted to confirm the protective effects of pomegranate seed oil (PSO) and black cumin oil (BCO) against α-A-induced hepatotoxicity. Rats exposed once to α-A (3 mg/kg bw, i.p.) or saline alone (0.1 ml, i.p.) were either left untreated or treated with PSO or BCO at a dose of 2 ml/kg bw/day by oral gavage on the same day, and the treatment was continued for 7 days. Serum aminotransferases (ALT and AST), alkaline phosphatase (ALP) and total protein levels were measured and the active caspase 3 (cl-caspase 3) was evaluated by western blotting in the liver. Serum ALT, AST and ALP levels tended to decrease in the α-A exposed group, but no statistically significant difference was found compared to the saline group (p > 0.05). PSO and BCO did not affect serum liver function tests in rats exposed to saline or α-A. α-A toxicity was demonstrated by a significant decrease in serum total protein level (p < 0.05), a significant increase in liver cl-caspase 3 expression (p < 0.05), and structural liver damage mainly characterized by mononuclear inflammation and steatosis. When α-A exposed rats were treated with BCO, the increase in cl-caspase 3 was not inhibited, on the contrary BCO increased cl-caspase 3 in healthy rats (p < 0.05). PSO significantly ameliorated α-A-induced cl-caspase 3 increase and inflammatory histopathology in the liver. Both PSO and BCO completely prevented α-A-induced protein degradation. The findings indicate that PSO and BCO may protect liver functions against α-A-induced hepatotoxicity, encouraging future comprehensive studies to test them at different doses and frequency.

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Amanita phalloides is one of the deadliest mushrooms worldwide, causing most fatal cases of mushroom poisoning. Among the poisonous substances of Amanita phalloides, amanitins are the most lethal toxins to humans. Currently, there are no specific antidotes available for managing amanitin poisoning and treatments are lack of efficacy. Amanitin mainly causes severe injuries to specific organs, such as the liver, stomach, and kidney, whereas the lung, heart, and brain are hardly affected. However, the molecular mechanism of this phenomenon remains not understood. To explore the possible mechanism of organ specificity of amanitin-induced toxicity, eight human cell lines derived from different organs were exposed to α, ß, and γ-amanitin at concentrations ranging from 0.3 to 100 µM. We found that the cytotoxicity of amanitin differs greatly in various cell lines, among which liver-derived HepG2, stomach-derived BGC-823, and kidney-derived HEK-293 cells are most sensitive. Further mechanistic study revealed that the variable cytotoxicity is mainly dependent on the different expression levels of the organic anion transporting polypeptide 1B3 (OATP1B3), which facilitates the internalization of amanitin into cells. Besides, knockdown of OATP1B3 in HepG2 cells prevented α-amanitin-induced cytotoxicity. These results indicated that OATP1B3 may be a crucial therapeutic target against amanitin-induced organ failure.

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Poisonous mushrooms containing α-amatoxin can be lethal, making it imperative to develop a rapid and sensitive detection method for α-amatoxin. Utilizing the DNA tetrahedral structure as its foundation, the aptamer allows controlled density and orientation. Consequently, we designed aptamer tetrahedral functionalized magnetic beads that specifically target α-amanitin to release complementary DNA (C-DNA) strands. These strands were then employed as primers to initiate rolling circle amplification (RCA) with fluorescent dyes. The combination of SYBR Green I detection probes facilitated the amplification of the detection signal, enhancing the detection sensitivity of the aptasensor. The calculated detection limit was determined to be 3 ng/mL, a magnitude lower than that of other aptasensors by 2 orders of magnitude. The aptasensor integrates the advantages of high sensitivity and specificity, offering a simple and reliable rapid detection method for α-amanitin analysis.

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Amanita phalloides is the primary species responsible for fatal mushroom poisoning, as its main toxin, α-amanitin, irreversibly and potently inhibits eukaryotic RNA polymerase II (RNAP II), leading to cell death. There is no specific antidote for α-amanitin, which hinders its clinical application. However, with the advancement of precision medicine in oncology, including the development of antibody-drug conjugates (ADCs), the potential value of various toxic small molecules has been explored. These ADCs ingeniously combine the targeting precision of antibodies with the cytotoxicity of small-molecule payloads to precisely kill tumor cells. We searched PubMed for studies in this area using these MeSH terms "Amanitins, Alpha-Amanitin, Therapeutic use, Immunotherapy, Immunoconjugates, Antibodies" and did not limit the time interval. Recent studies have conducted preclinical experiments on ADCs based on α-amanitin, showing promising therapeutic effects and good tolerance in primates. The current challenges include the not fully understood toxicological mechanism of α-amanitin and the lack of clinical studies to evaluate the therapeutic efficacy of ADCs developed based on α-amanitin. In this article, we will discuss the role and therapeutic efficacy of α-amanitin as an effective payload in ADCs for the treatment of various cancers, providing background information for the research and application strategies of current and future drugs.

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Amatoxins are responsible for most fatal mushroom poisoning cases, as it causes both hepatotoxicity and nephrotoxicity. However, studies on amatoxin nephrotoxicity are limited. Here, we investigated nephrotoxicity over 4 days and nephrotoxicity/hepatotoxicity over 14 days in mice. The organ weight ratio, serological indices, and tissue histology results indicated that a nephrotoxicity mouse model was established with two stages: (1) no apparent effects within 24 h; and (2) the appearance of adverse effects, with gradual worsening within 2-14 days. For each stage, the kidney transcriptome revealed patterns of differential mRNA expression and significant pathway changes, and Western blot analysis verified the expression of key proteins. Amanitin-induced nephrotoxicity was directly related to RNA polymerase II because mRNA levels decreased, RNA polymerase II-related pathways were significantly enriched at the transcription level, and RNA polymerase II protein was degraded in the early poisoning stage. In the late stage, nephrotoxicity was more severe than hepatotoxicity. This is likely associated with inflammation because inflammation-related pathways were significantly enriched and NF-κB activation was increased in the kidney.

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Mushroom poisonings caused by Amanita phalloides are the leading cause of mushroom-related deaths worldwide. Alpha-Amanitin (α-AMA), a toxic substance present in these mushrooms, is responsible for the resulting hepatotoxicity and nephrotoxicity. The objective of our study was to determine the distribution of α-AMA in Balb/c mice by labeling with Iodine-131. Mice were injected with a toxic dose (1.4 mg/kg) of α-AMA labeled with Iodine-131. The mice were sacrificed at the 1st, 2nd, 4th, 8th, 24th, and 48th hours under anesthesia. The organs of the mice were removed, and their biodistribution was assessed in all experiments. The percent injected dose per gram (ID/g %) value for kidney, liver, lung, and heart tissues at 1st hour were 1.59 ± 0.07, 1.25 ± 0.33, 3.67 ± 0.80 and 1.07 ± 0.01 respectively. This study provides insights into the potential long-term effects of α-AMA accumulation in specific organs. Additionally, this study has generated essential data that can be used to demonstrate the impact of antidotes on the biological distribution of α-AMA in future toxicity models.

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Wild mushroom poisoning is a global public health concern, with mushrooms containing amatoxins being the main cause of fatalities. Mushrooms from the genus Amanita and Galerina contain amatoxins. Here we present a case of wild mushroom poisoning that affected three individuals, resulting in two fatalities. Within 10-15 hours after consumption, they experienced symptoms of gastroenteritis such as vomiting, abdominal pain, and diarrhea. One individual sought medical attention promptly and recovered, while the other two sought medical help nearly two or three days after the onset of symptoms, by which time their conditions had already worsened and led to their deaths. The mushrooms were identified belonging to genus Galerina, and laboratory test revealed variations in toxin levels among mushrooms collected from different parts of the decaying stump. The higher levels of α-amanitin, ß-amanitin, and γ-amanitin were detected near the base of the tree stump, but trace levels of α-amanitin were found near the top of the stump, while ß-amanitin and γ-amanitin were undetectable. This case emphasizes the importance of seeking immediate medical attention when experiencing delayed-onset gastrointestinal symptoms, as it may indicate more severe mushroom poisoning, particularly amatoxin poisoning. Timely and appropriate treatment is equally important. Additionally, consuming different units of the mushrooms in the same incident can lead to varying prognoses due to differences in toxin levels.

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What is already known about this topic?: Fatal poisonings caused by wild mushrooms containing amanita toxins pose a significant threat in the southern regions of China. These toxins primarily induce gastrointestinal symptoms initially, which are then followed by potentially life-threatening acute liver damage. What is added by this report?: This report contributes to the existing knowledge on these cases of poisoning by documenting the second occurrences in Hebei Province and the first occurrences in Xingtai City. Five individuals reported consuming wild mushrooms from the same origin, and laboratory tests confirmed the presence of α-amanitin in their blood samples. What are the implications for public health practice?: This underscores the risk associated with the collection and consumption of amanita toxin-containing mushrooms in Hebei. It is important to note that the identification of toxic and non-toxic mushrooms should not solely rely on personal experience or appearance.

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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 ß-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or ß-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. ß-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.

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OBJECTIVE To study the toxicokinetics and tissue distribution characteristics of alpha-amanitin in rats.METHODS The tail venous blood was collected from SD rats before and 5,10,20,30 and 45 min,1,1.5,2.5,4 and 8 h after intraperitoneal injection of alpha-amanitin(1.5 mg·kg-1),and the concentration of alpha-amanitin in blood was determined by liquid chromatography-mass spectrometry(LC-MS/MS).DAS 2.0 software was used to analyze and plot the drug-time curve with toxicokinetic parame-ters.Based on the toxicokinetics results,18 SD rats were randomly divided into three groups.The rats were sacrificed,and left ventricular arterial(LVA)blood and 9 types of tissue samples involving the heart,liver,spleen,lung,kidney,whole brain,small intestine,stomach wall and testis were collected 15 min,40 min and 2.5 h after dosing,and the concentrations of alpha-amanitin were measured by LC-MS/MS to obtain the tissue distribution results of alpha-amanitin in SD rats.RESULTS Toxicokinetics studies revealed that the peak blood concentration(Cmax)was(633±121)μg·L-1,the elimination half-life(T1/2)was(0.72±0.37)h,and the peak time(Tmax)was(0.52±0.16)h.The total clearance rate(CLz)was(1.62±0.26)L·h·kg-1,the area under the curve(AUC0-t)was(946±183)μg·h·L-1,and the mean reten-tion time(MRT0-t)was(1.18±0.17)h.The apparent volume of distribution(Vz)was(1.65±0.86)L·kg-1.The results of tissue distribution study showed that alpha-amanitin was widely distributed in SD rats with the highest concentration in the kidney,followed by the lung,small intestines,stomach wall,LVA blood and liver,but was low in the heart,spleen,testicles and other tissues,and very low in the brain.Alpha-amanitin was absorbed and eliminated quickly,peaked at 40 min in each tissue,and the concen-tration was minimized after 2.5 h.CONCLUSION The absorption and elimination of alpha-amanitin by intraperitoneal injection are rapid in SD rats,and the blood concentration reaches the peak about 31 min after administration,but can not be detected 4 h later.Alpha-amanitin is mainly distributed in the kidney,followed by the tissues and metabolic organs with rich blood flow,such as the lung,small intestines,stomach wall,LVA blood and liver.The content of alpha-amanitin is low in the heart,spleen,testicles and other tissues,and very low in the brain.It is speculated that it may have toxic targeting effect on the kidney and low blood-brain barrier permeability.

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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.

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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.

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α-Amanitin, the primary lethal toxin of Amanita, specifically targets the liver, causing oxidative stress, hepatocyte apoptosis, and irreversible liver damage. As little as 0.1 mg/kg of α-amanitin can be lethal for humans, and there is currently no effective antidote for α-amanitin poisoning. Cannabidiol is a non-psychoactive natural compound derived from Cannabis sativa that exhibits a wide range of anti-inflammatory, antioxidant, and anti-apoptotic effects. It may play a protective role in preventing liver damage induced by α-amanitin. To investigate the potential protective effects of cannabidiol on α-amanitin-induced hepatocyte apoptosis and oxidative stress, we established α-amanitin exposure models using C57BL/6J mice and L-02 cells in vitro. Our results showed that α-amanitin exposure led to oxidative stress, apoptosis, and DNA damage in both mouse hepatocytes and L-02 cells, resulting in the death of mice. We also found that cannabidiol upregulated the level of Nrf2 and antioxidant enzymes, alleviating apoptosis, and oxidative stress in mouse hepatocytes and L-02 cells and increasing the survival rate of mice. Our findings suggest that cannabidiol has hepatoprotective effects through the regulation of Nrf2 and antioxidant enzymes and may be a potential therapeutic drug for Amanita poisoning.

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The misuse of poisonous mushrooms containing amatoxins causes acute liver failure (ALF) in patients and is a cause of significant mortality. Although the toxic mechanisms of α-amanitin (α-AMA) and its interactions with RNA polymerase II (RNAP II) have been studied, α-AMA effector proteins that can interact with α-AMA in hepatocytes have not been systematically studied. Limited proteolysis-coupled mass spectrometry (LiP-MS) is an advanced technology that can quickly identify protein-ligand interactions based on global comparative proteomics. This study identified the α-AMA effector proteins found in human hepatocytes, following the detection of conformotypic peptides using LiP-MS coupled with tandem mass tag (TMT) technology. Proteins that are classified into protein processing in the endoplasmic reticulum and the ribosome during the KEGG pathway can be identified through affinity evaluation, according to α-AMA concentration-dependent LiP-MS and LiP-MS in hepatocytes derived from humans and mice, respectively. The possibility of interaction between α-AMA and proteins containing conformotypic peptides was evaluated through molecular docking studies. The results of this study suggest a novel path for α-AMA to induce hepatotoxicity through interactions with various proteins involved in protein synthesis, as well as with RNAP II.

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