Amanitin-induced variable cytotoxicity in various cell lines is mediated by the different expression levels of OATP1B3.

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.

Kidney toxicity and transcriptome analyses of male ICR mice acutely exposed to the mushroom toxin α-amanitin.

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.

Biodistribution of radiolabeled alpha-amanitin in mice: An Investigation.

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.

Unraveling Hematotoxicity of α-Amanitin in Cultured Hematopoietic Cells.

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.

Identification of α-amanitin effector proteins in hepatocytes by limited proteolysis-coupled mass spectrometry.

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.

Rapid detection of α-amanitin and ß-amanitin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study of Lepiota brunneoincarnata.

PURPOSE: Lepiota brunneoincarnata is a well-known poisonous mushroom and is responsible for fatal mushroom poisoning cases worldwide. α-Amanitin and ß-amanitin are the main amatoxin compounds of Lepiota brunneoincarnata. However, there are no published toxicokinetic studies of Lepiota brunneoincarnata. To study the toxicokinetics of Lepiota brunneoincarnata, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of α-amanitin and ß-amanitin in rat plasma. METHODS: UPLC-MS/MS analyses were performed with a triple quadrupole mass spectrometer in positive-ion mode. The sensitivity of α-amanitin and ß-amanitin detection was increased by inhibiting the production of [M + Na]+ adducts. α-Amanitin and ß-amanitin were separated and quantified on an UPLC octadecyl silyl column in only 2.5 min. RESULTS: The linear ranges were 3.0-3000 ng/mL for α-amanitin and 1.8-1800 ng/mL for ß-amanitin with a correlation coefficient r > 0.99 for both analytes. The lower limit of quantification of 3.0 ng/mL for α-amanitin and 1.8 ng/mL for ß-amanitin was achieved using only 50 µL of rat plasma. The accuracy of α-amanitin and ß-amanitin was between - 9.5 and 7.0% with the precision ranged from 2.2 to 12.5%. The developed method was then applied for Lepiota brunneoincarnata toxicokinetic study after intravenous administration of Lepiota brunneoincarnata extracts. CONCLUSIONS: Establishing UPLC-MS/MS method for quantifying amanitines in rat plasma successfully enabled toxicokinetic study of Lepiota brunneoincarnata extracts.

Antidotal effect of cyclosporine A against α-amanitin toxicity in CD-1 mice, at clinical relevant doses.

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 after it 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 cyclosporine A i.p. at 8 and 12 h post α-amanitin) resulted in the full survival of α-amanitin-intoxicated mice, up to 30 days after the toxin's administration. Since α-amanitin is a substrate of the organic-anion-transporting polypeptide 1B3 and cyclosporine A inhibits this transporter and is 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 advocates for its use at an early stage of α-amanitin intoxications.

Erdosteine reduces cytotoxicity induced by alpha- and beta-amanitin, but not gamma-amanitin, in CA3 hepatocyte cultures.

Amanitin poisoning still has no particular, effective antidote. Erdosteine has been shown to protect numerous tissues, particularly those in the liver. This study investigates the potential therapeutic effects of erdosteine on alpha-, beta- and gamma-amanitin-induced hepatotoxicity in in vitro models. Three hours after administering amatoxins at various concentrations (1-50 µg/mL) to the cells of the C3A human hepatocyte cell line, erdosteine was administered in different concentrations (i.e., 1, 10, 50, 100 and 250 µg/mL). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was selected to determine cell viability. When concentrations of 1, 10, 50, 100 and 250 µg/mL of erdosteine were applied to cell lines, the following cell viability rates were obtained: 106%,99%,93%,86% and 86%, respectively, at a 10 µg/mL alpha-amanitin-induced toxicity; 43%,41%,41%,37% and 35%, respectively, at a 25 µg/mL alpha-amanitin-induced toxicity; 44%,42%,41%,39% and 41%, respectively, at a 50 µg/mL alpha-amanitin-induced toxicity; 136%,142%,143%,137% and 120%, respectively, at a 10 µg/mL beta-amanitin-induced toxicity; 113%,107%,107%,106% and 86%, respectively, at a 25 µg/mL beta-amanitin-induced toxicity; 78%,77%,77%,74% and 70%, respectively, at a 10 µg/mL gamma-amanitin-induced toxicity; and 39%,40%,39%,35% and 31%, respectively, at a 25 µg/mL gamma-amanitin-induced toxicity. This study was the first to evaluate the in vitro efficacy of erdosteine in cytotoxicity induced by alpha-, beta- and gamma-amanitin. Non-high (low and medium) doses of erdosteine are capable of nearly entirely preventing toxicity at mild hepatotoxic concentrations caused by amatoxin and partially preventing toxicity at moderate and severe concentrations. The beneficial effects of erdosteine, especially on the toxicity of alpha- and beta-amanitin, are promising.

Autophagy Promotes α-Amanitin-Induced Apoptosis of Hepa1-6 Liver Cells.

It is estimated that 90% of deaths from food poisoning in China can be attributed to Amanita poisoning, whose main toxin is α-amanitin. Studies showed that apoptosis plays a critical role in liver injuries induced by α-amanitin. Although the relationship between autophagy and apoptosis in different liver models has been addressed many times, whether autophagy plays a pro or con effect on α-amanitin-induced apoptosis has not been clarified. Therefore, this study was conducted to explore the effect of autophagy in α-amanitin-induced apoptosis in Hepa1-6 liver cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was applied to determine cell viability, a 2',7'-dichlorofluorescin diacetate probe was used to monitor reactive oxygen species (ROS) levels, a flow cytometer and dansylcadaverine (MDC) staining were used to observe α-amanitin-induced apoptosis and autophagy, respectively, and apoptosis and autophagy proteins were assessed by western blotting. The results showed that α-amanitin suppressed cell viability in a time- and concentration-dependent manner. Moreover, the release of ROS was increased with increasing α-amanitin amount. Cell apoptosis and autophagy were noticed and characterized by the increased apoptosis rate and autophagic vesicles under a fluorescence microscope as well as upregulation of Bax/Bcl-2, cleaved caspase-3, and LC3-II/I and downregulation of p62. Further, the autophagy activator rapamycin (Rap) and the inhibitor 3-methylademine (3-MA) were introduced, which showed that the apoptosis rate and the ratio of Bax/Bcl-2 as well as the protein expression level of cleaved caspase-3 increased significantly with the pretreatment of Rap and decreased remarkably with the pretreatment of 3-MA. Moreover, cell viability was found to decrease further with the promotion of autophagy. Notably, the ROS level was attenuated after autophagy was elevated. In conclusion, autophagy could promote α-amanitin-induced Hepa1-6 cell apoptosis, and the process is unassociated with ROS levels. This research provides a theoretical basis for the study of the toxicological mechanism of α-amanitin-induced liver injuries.

Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro.

Alpha-amanitin (α-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography-tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Toxin components and toxicological importance of Galerina marginata from Turkey.

Amatoxins, most of which are hepatotoxic, can cause fatal intoxication. While mushrooms in the amatoxin-containing Galerina genus are rare, they can poison humans and animals worldwide. Few studies have profiled the toxicity of Galerina marginata. In addition, many studies indicate that macrofungi can have different characteristics in different regions. In this study, the quantities of toxins present in G. marginata from different provinces in Turkey were analysed using reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). G. marginata samples were collected from three different regions of Turkey. The taxonomic categorization of mushrooms was based on their micro- and macroscopic characteristics. The presence of toxins α-amanitin (AA), ß-amanitin (BA), γ-amanitin (GA), phalloidin (PHD) and phallacidin (PHC) quantities were measured using RP-HPLC-UV and then were confirmed using LC-ESI-MS/MS. BA levels were higher than AA levels in G. marginata mushrooms collected from all three regions. Moreover, the levels of GA were below the detection limit and no phallotoxins were detected. This is the first study to identify and test the toxicity of G. marginata collected from three different regions of Turkey using RP-HPLC-UV. This is also the first study to confirm the UV absorption of amatoxins in G. marginata using LC-ESI-MS/MS, which is a far more sensitive process. More studies evaluating the toxicity of G. marginata in other geographic regions of the world are needed.

Assessment of α-amanitin toxicity and effects of silibinin and penicillin in different in vitro models.

Silibinin (Sil) is used as hepatoprotective drug and is approved for therapeutic use in amanitin poisoning. In our study we compared Sil-bis-succinate (SilBS), a water-soluble drug approved for i.v.-administration, with Sil solved in ethanol (SilEtOH), which is normally used in research. We challenged monocultures or 3D-microtissues consisting of HepG2 cells or primary hepatocytes with α-amanitin and treated with SILBS, SILEtOH, penicillin and combinations thereof. Cell viability and the integrity of the microtissues was monitored. Finally, the expression of the transporters OATP1B1 and B3 was analyzed by qRT-PCR. We demonstrated that primary hepatocytes were more sensitive to α-amanitin compared to HepG2. Primary hepatocytes cultures were protected by SilBS and SilEtOH independent of penicillin from the cytotoxic effects of α-amanitin. Subsequent studies of the expression profile of the transporters OATP1B1/B3 revealed that primary hepatocytes do express both whereas in HepG2 cells they were hardly detectable. Our study showed that SilBS has significant advantage over SilEtOH with no additional benefit of penicillin. Moreover, HepG2 cells may not represent an appropriate model to investigate Amanita phalloides poisoning in vitro with focus on OATP transporters since these cells are lacking sensitivity towards α-amanitin probably due to missing cytotoxicity-associated transporters suggesting that primary hepatocytes should be preferred in this context.

In vitro mechanistic studies on α-amanitin and its putative antidotes.

α-Amanitin plays a key role in Amanita phalloides intoxications. The liver is a major target of α-amanitin toxicity, and while RNA polymerase II (RNA Pol II) transcription inhibition is a well-acknowledged mechanism of α-amanitin toxicity, other possible toxicological pathways remain to be elucidated. This study aimed to assess the mechanisms of α-amanitin hepatotoxicity in HepG2 cells. The putative protective effects of postulated antidotes were also tested in this cell model and in permeabilized HeLa cells. α-Amanitin (0.1-20 µM) displayed time- and concentration-dependent cytotoxicity, when evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red uptake assays. Additionally, α-amanitin decreased nascent RNA synthesis in a concentration- and time-dependent manner. While α-amanitin did not induce changes in mitochondrial membrane potential, it caused a significant increase in intracellular ATP levels, which was not prevented by incubation with oligomycin, an ATP synthetase inhibitor. Concerning the cell redox status, α-amanitin did not increase reactive species production, but caused a significant increase in total and reduced glutathione, which was abolished by pre-incubation with the inhibitor of gamma-glutamylcysteine synthase, buthionine sulfoximine. None of the tested antidotes [N-acetyl cysteine, silibinin, benzylpenicillin, and polymyxin B (PolB)] conferred any protection against α-amanitin-induced cytotoxicity in HepG2 cells or reversed the inhibition of nascent RNA caused by the toxin in permeabilized HeLa cells. Still, PolB interfered with RNA Pol II activity at high concentrations, though not impacting on α-amanitin observed cytotoxicity. New hepatotoxic mechanisms of α-amanitin were described herein, but the lack of protection observed in clinically used antidotes may reflect the lack of knowledge on their true protection mechanisms and may explain their relatively low clinical efficacy.

Effects of resveratrol on alpha-amanitin-induced nephrotoxicity in BALB/c mice.

Alpha-amanitin (α-AMA), the primary toxin of Amanita phalloides, is known to cause nephrotoxicity and hepatotoxicity. Resveratrol is an antioxidant that has shown efficacy in many nephrotoxicity models. The aim of this study was to investigate the effects of resveratrol against the early and late stages of α-AMA-induced nephrotoxicity, compared to those of silibinin, a well-known antidote for poisoning by α-AMA-containing mushrooms. Mice kidney tissues were obtained from five groups: (1) α-AMA + NS (simultaneous administration of α-AMA and normal saline), (2) α-AMA + SR (simultaneous administration of α-AMA and resveratrol), (3) α-AMA + 12R (resveratrol administration 12 h after α-AMA administration), (4) α-AMA + 24R (resveratrol administration 24 h after α-AMA administration), and (5) α-AMA + Sil (simultaneous administration of α-AMA and silibinin). Histomorphological and biochemical analyses were performed to evaluate kidney damage and oxidant-antioxidant status in the kidney. Scores of renal histomorphological damage decreased significantly in the early resveratrol treatment groups (α-AMA + SR and α-AMA + 12R), compared to those in the α-AMA + NS group (p < 0.05). Catalase levels increased significantly in the α-AMA + SR group, compared to those in the α-AMA + NS group (p < 0.001). Early resveratrol administration within 12 h after α-AMA ingestion may reverse the effects of α-AMA-induced nephrotoxicity, partly through its antioxidant action, thereby suggesting its potential as a treatment for poisoning by α-AMA-containing mushrooms.

Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity.

Amanitin-induced apoptosis is proposed to have a significant effect on the pathogenesis of liver damage. However, few reports have focused on proteome changes induced by α-amanitin (α-AMA). Here, we evaluated changes in mitochondrial proteins of hepatocytes in response to 2 µM α-AMA, a concentration at which α-AMA-induced cell damage could be rescued at cellular level by common clinical drugs. We found 56 proteins were differentially expressed in an α-AMA-treated group. Among them, 38 proteins were downregulated and 18 were upregulated. Downregulated functional proteins included importer TOMM40, respiratory chain component cytochrome C, and metabolic enzymes of citrate acid cycle such as malate dehydrogenase, which localize on the mitochondrial outer membrane, inner membrane and matrix respectively. Immunoblot analysis showed that α-AMA decreased mitochondrial import receptor subunit TOMM40 and cytochrome c accompanied by an increase in the cytosol although their total protein levels were not affected significantly. The mitochondrial membrane potential was also destroyed by α-AMA and was restored by the clinical drug silibinin. Immunofluorescence suggested that mitochondrial morphology did not change. Taken together, our results provide further insights into the toxic mechanism of α-AMA on hepatocytes.

A Comparison of the Effectiveness of Silibinin and Resveratrol in Preventing Alpha-Amanitin-Induced Hepatotoxicity.

Amanita phalloides species mushrooms containing alpha-amanitin (α-AMA) are responsible for the majority of fatal mushroom intoxications and can lead to severe poisonings resulting in hepatotoxicity and acute hepatic failure. Existing antidotes, such as silibinin, are not sufficiently effective in the prevention and/or resolution of α-AMA-induced hepatotoxicity. We investigated the effects of resveratrol on α-AMA-induced hepatotoxicity and compared with silibinin, a known antidote using in vivo and in vitro toxicity models. In the in vivo protocol, resveratrol (30 mg/kg) was given simultaneously with α-AMA (α-AMA + SR) or 12 (α-AMA + 12R) or 24 (α-AMA + 24R) hr after α-AMA administration. Silibinin (5 mg/kg) (α-AMA + Sil) and normal saline (α-AMA + NS) were given simultaneously with α-AMA. We found that liver transaminase levels in α-AMA + SR and α-AMA + 12R groups and histomorphologic injury score in the α-AMA + SR, α-AMA + 12R, α-AMA + 24R and α-AMA + Sil groups were significantly lower than that of the α-AMA + NS group. Resveratrol decreased mononuclear cell infiltration, necrosis and active caspase-3 immunopositivity in the liver. In the in vitro protocol, the effects of resveratrol and silibinin were evaluated in a reduction in cell viability induced by α-AMA in THLE-2 and THLE-3 hepatocytes. Neither resveratrol nor silibinin was found to be effective in increasing cell viability decreased by α-AMA + NS. As a conclusion, resveratrol was found to be effective in α-AMA-induced hepatotoxicity with its anti-inflammatory properties in in vivo conditions. It is a promising compound with the potential for use in the treatment of hepatotoxicity associated with Amanita phalloides type mushroom poisonings.

A cost-effective LC-MS/MS method for identification and quantification of α-amanitin in rat plasma: Application to toxicokinetic study.

α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.

Evaluation of the genotoxicity of alpha-amanitin in mice bone marrow cells.

Alpha-amanitin is a known cytotoxic substance found in some mushroom species including Amanita phalloides. Its main mechanism of action is to block the transcription, which can lead to cell death. Lack of reports on the genotoxicity of this toxin was an inspiration for undertaking this experiment. Genotoxic effect of α-amanitin on balb/c mice bone marrow cells was tested using: comet assay and chromosomal aberration test. The tested substance was given once by intraperitoneal administration to animals at doses: 0.1 mg/kg, 0.15 mg/kg and 0.25 mg/kg (LD50) body weight with 48 h exposure. The comet assay demonstrated a statistically significant increase in DNA damage for all the investigated α-amanitin doses compared to the negative control (p < 0.0001). The exposure to 0.15 and 0.25 mg/kg doses of α-amanitin also generated a statistically significant increase in the frequency of chromosomal aberrations in bone marrow cells of mice compared to the negative control (p < 0.05). The genotoxic effect induced by α-amanitin in mammalian cells can result in genome instability and its functional consequences.

The role of oxidative stress in α-amanitin-induced hepatotoxicityin an experimental mouse model.

BACKGROUND/AIM: This study aimed to evaluate oxidative stress markers of liver tissue in a mouse α-amanitin poisoning model with three different toxin levels. MATERIALS AND METHODS: The mice were randomly divided into Group 1 (control), Group 2 (0.2 mg/kg), Group 3 (0.6 mg/kg), and Group 4 (1.0 mg/kg). The toxin was injected intraperitoneally and 48 h of follow-up was performed before sacrifice. RESULTS: Median superoxide dismutase activities of liver tissue in Groups 3 and 4 were significantly higher than in Group 1 (for both, P = 0.001). The catalase activity in Group 2 was significantly higher, but in Groups 3 and 4 it was significantly lower than in Group 1 (for all, P = 0.001). The glutathione peroxidase activities in Groups 2, 3, and 4 were significantly higher than in Group 1 (P = 0.006, P = 0.001, and P = 0.001, respectively). The malondialdehyde levels of Groups 3 and 4 were significantly higher than Group 1 (P = 0.015 and P = 0.003, respectively). The catalase activity had significant correlations with total antioxidant status and total oxidant status levels (r = 0.935 and r = -0.789, respectively; for both, P < 0.001). CONCLUSION: Our findings support a significant role for increased oxidative stress in α-amanitin-induced hepatotoxicity.

Hepatoprotective Effects and Mechanisms of Action of Triterpenoids from Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) on α-Amanitin-Induced Liver Injury in Mice.

Most fatal mushroom poisonings are caused by species of the genus Amanita; the amatoxins are responsible for acute liver failure and death in humans. Ganoderma lucidum is a well-known traditional medicinal mushroom that has been shown to have obvious hepatoprotective effects. This study evaluated the hepatoprotective effects of triterpenoids from G. lucidum on liver injury induced by a-amanitin (α-AMA) in mice and the mechanisms of action of these triterpenoids, including radical scavenging and antiapoptosis activities. Mice were treated with α-AMA, followed by G. lucidum total triterpenoids or individual triterpenoids, and their hepatoprotective effects were compared with those of the reference drug silibinin (SIL). Treatment with SIL, G. lucidum total triterpenoids, and each of the 5 individual triterpenoids significantly reduced serum alanine aminotransaminase and aspartate ami- notransaminase concentrations and reduced mortality rates 20-40%. Moreover, triterpenoids and SIL significantly enhanced superoxide dismutase and catalase activity and reduced malondialdehyde content in livers. Treatment with ganoderic acid C2 significantly inhibited DNA fragmentation and decreased caspase-3, -8, and -9 activities. The results demonstrated that triterpenoids have hepatoprotective effects on α-AMA-induced liver injury and that their hepatoprotective mechanisms may be the result of their antioxidative and radical scavenging activities and their inhibition of apoptosis.