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.

Rationally Designed Amanitins Achieve Enhanced Cytotoxicity.

For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure-activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.

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.

Toxic Effects of Amanitins: Repurposing Toxicities toward New Therapeutics.

The consumption of mushrooms has become increasingly popular, partly due to their nutritional and medicinal properties. This has increased the risk of confusion during picking, and thus of intoxication. In France, about 1300 cases of intoxication are observed each year, with deaths being mostly attributed to Amanita phalloides poisoning. Among amatoxins, α- and ß-amanitins are the most widely studied toxins. Hepatotoxicity is the hallmark of these compounds, leading to hepatocellular failure within three days of ingestion. The toxic mechanisms of action mainly include RNA polymerase II inhibition and oxidative stress generation, leading to hepatic cell apoptosis or necrosis depending on the doses ingested. Currently, there is no international consensus concerning Amanita phalloides poisoning management. However, antidotes with antioxidant properties remain the most effective therapeutics to date suggesting the predominant role of oxidative stress in the pathophysiology. The partially elucidated mechanisms of action may reveal a suitable target for the development of an antidote. The aim of this review is to present an overview of the knowledge on amanitins, including the latest advances that could allow the proposal of new innovative and effective therapeutics.

Intravenous rifampicin use in the management of amanita phalloides toxicity.

Context: Amanita phalloides related toxicity from amatoxins can result in acute liver and multi-organ failure and is responsible for 90% of all mushroom poisoning death. However, more evidence is needed in regards to different management strategies.Case details: We present two cases of amanita mushroom ingestion who were treated with intravenous rifampicin.Discussion: Further study is needed to establish the efficacy and role of rifampicin in amatoxin related mushroom poisoning.

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.

Simultaneous identification and characterization of amanita toxins using liquid chromatography-photodiode array detection-ion trap and time-of-flight mass spectrometry and its applications.

Rapid and accurate identification of multiple toxins for clinical diagnosis and treatment of mushroom poisoning cases is still a challenge, especially with the lack of authentic references. In this study, we developed an effective method for simultaneous identification of amanita peptide toxins by liquid chromatography coupled with photodiode array detection and ion trap time-of-flight mass spectrometry. The accuracy and selectivity of the methodology were validated through similar multiple fragmentation patterns and characteristic ions of standard α- and ß-amanitin. The developed method could successfully separate and identify major toxic constituents in Amanita mushrooms. Two amatoxins and three phallotoxins were confirmed in a single run through their fragmentation patterns and characteristic ions, which can be used as diagnostic fragment ions to identify mushroom toxins in complex samples. Furthermore, the performance of the developed method was verified by using real biological samples, including plasma and urine samples collected from rats after intraperitoneal administration of toxins. Thus, the development methodology could be crucial for the accurate detection of mushroom toxins without standard references.

Circulating monocytes accelerate acute liver failure by IL-6 secretion in monkey.

Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin-induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL-6 increase was the most rapid and drastic. Interestingly, we found that IL-6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte-derived IL-6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.

A universal method for the identification of genes encoding amatoxins and phallotoxins in poisonous mushrooms

Background: As the currently known diagnostic DNA targets amplified in the PCR assays for detection of poisonous mushrooms have their counterparts in edible species, there is a need to design PCR primers specific to the genes encoding amanitins and phallotoxins, which occur only in poisonous mushrooms. Objective: The aim of the study was testing of PCR-based method for detection of all genes encoding hepatotoxic cyclic peptides - amanitins and phallotoxins present in the most dangerous poisonous mushrooms. Material and Methods: Degenerate primers in the PCR were designed on the basis of amanitins (n=13) and phallotoxins (n=5) genes in 18 species of poisonous mushrooms deposited to Genbank of the National Center for Biotechnology Information. Results: The specificity of the PCR assays was confirmed against 9 species of edible mushrooms, death cap - Amanita phalloides and panther cap - Amanita pantherina. Conclusions: Designed two couples of PCR-primers specific to amanitins and phallotoxins genes can be recommended for detection of Amanita phalloides and other mushroom species producing hepatotoxic cyclic peptides - amanitins and phallotoxins.

Cyclopeptide toxins of lethal amanitas: Compositions, distribution and phylogenetic implication.

Lethal amanitas (Amanita sect. Phalloideae) are responsible for 90% of all fatal mushroom poisonings. Since 2000, more than ten new lethal Amanita species have been discovered and some of them had caused severe mushroom poisonings in China. However, the contents and distribution of cyclopeptides in these lethal mushrooms remain poorly known. In this study, the diversity of major cyclopeptide toxins in seven Amanita species from Eastern Asia and three species from Europe and North America were systematically analyzed, and a new approach to inferring phylogenetic relationships using cyclopeptide profile was evaluated for the first time. The results showed that there were diversities of the cyclopeptides among lethal Amanita species, and cyclopeptides from Amanita rimosa and Amanita fuligineoides were reported for the first time. The amounts of amatoxins in East Asian Amanita species were significantly higher than those in European and North American species. The analysis of distribution of amatoxins and phallotoxins in various Amanita species demonstrated that the content of phallotoxins was higher than that of amatoxins in Amanita phalloides and Amanita virosa. In contrast, the content of phallotoxins was significantly lower than that of amatoxins in all East Asian lethal Amanita species tested. However, the distribution of amatoxins and phallotoxins in different tissues showed the same tendency. Eight cyclopeptides and three unknown compounds were identified using cyclopeptide standards and high-resolution MS. Based on the cyclopeptide profiles, phylogenetic relationships of lethal amanitas were inferred through a dendrogram generated by UPGMA method. The results showed high similarity to the phylogeny established previously based on the multi-locus DNA sequences.

[Forensic medical diagnostics of intoxication with certain poisonous mushrooms in the case of the lethal outcome in a hospital].

The present study was undertaken with a view to improving forensic medical diagnostics of intoxication with poisonous mushrooms in the cases of patients' death in a hospital. A total of 15 protocols of forensic medical examination of the corpses of the people who had died from acute poisoning were available for the analysis. The deathly toxins were amanitin and muscarine contained in various combinations in the death cap (Amanita phalloides) and the early false morels (Gyromitra esculenta and G. gigas). The main poisoning season in the former case was May and in the latter case August and September (93.4%). The mortality rate in the case of group intoxication (such cases accounted for 40% of the total) amounted to 28.6%. 40% of the deceased subjects consumed mushrooms together with alcohol. The poisoning caused the development of either phalloidin- or gyromitrin-intoxication syndromes (after consumption of Amanita phalloides and Gyromitra esculenta respectively). It is emphasized that the forensic medical experts must substantiate the diagnosis of poisoning with mushroom toxins based on the results of the chemical-toxicological and/or forensic chemical investigations. The relevant materials taken from the victim or the corpse should be dispatched for analysis not only within the first day but also on days 2-4 after intoxication. The mycological and genetic analysis must include the detection and identification of mushroom microparticles and spores in the smears from the oral cavity, vomiting matter, wash water, gastric and intestinal contents. In addition, the macro- and microscopic morphological signs, clinical data (major syndromes, results of laboratory studies, methods of treatment) should be taken into consideration as well as the time (season) of mushroom gathering, simultaneous poisoning in a group of people, and other pertinent information.

Acute liver injury and acute liver failure from mushroom poisoning in North America.

BACKGROUND & AIMS: Published estimates of survival associated with mushroom (amatoxin)-induced acute liver failure (ALF) and injury (ALI) with and without liver transplant (LT) are highly variable. We aimed to determine the 21-day survival associated with amatoxin-induced ALI (A-ALI) and ALF (A-ALF) and review use of targeted therapies. METHODS: Cohort study of all A-ALI/A-ALF patients enrolled in the US ALFSG registry between 01/1998 and 12/2014. RESULTS: Of the 2224 subjects in the registry, 18 (0.8%) had A-ALF (n = 13) or A-ALI (n = 5). At admission, ALF patients had higher lactate levels (5.2 vs. 2.2 mm, P = 0.06) compared to ALI patients, but INR (2.8 vs. 2.2), bilirubin (87 vs. 26 µm) and MELD scores (28 vs. 24) were similar (P > 0.2 for all). Of the 13 patients with ALF, six survived without LT (46%), five survived with LT (39%) and two died without LT (15%). Of the five patients with ALI, four (80%) recovered and one (20%) survived post-LT. Comparing those who died/received LT (non-spontaneous survivors [NSS]) with spontaneous survivors (SS), N-acetylcysteine was used in nearly all patients (NSS 88% vs. SS 80%); whereas, silibinin (25% vs. 50%), penicillin (50% vs. 25%) and nasobiliary drainage (0 vs. 10%) were used less frequently (P > 0.15 for all therapies). CONCLUSION: Patients with mushroom poisoning with ALI have favourable survival, while around half of those presenting with ALF may eventually require LT. Further study is needed to define optimal management (including the use of targeted therapies) to improve survival, particularly in the absence of LT.

Co-ingestion of amatoxins and isoxazoles-containing mushrooms and successful treatment: A case report.

Mushroom poisonings occur when ingestion of wild mushrooms containing toxins takes place, placing the consumers at life-threatening risk. In the present case report, an unusual multiple poisoning with isoxazoles- and amatoxins-containing mushrooms in a context of altered mental state and poorly controlled hypertension is presented. A 68-year-old female was presented to São João hospital (Portugal) with complaints of extreme dizziness, hallucinations, vertigo and imbalance, 3 h after consuming a stew of wild mushrooms. The first observations revealed altered mental state and elevated blood pressure. The examination of cooked mushroom fragments allowed a preliminary identification of Amanita pantherina. Gas chromatography-mass spectrometry (GC-MS) showed the presence of muscimol in urine. Moreover, through high-performance liquid chromatography-ultraviolet detection (HPLC-UV) analysis of the gastric juice, the presence of α-amanitin was found, showing that amatoxins-containing mushrooms were also included in the stew. After 4 days of supportive treatment, activated charcoal, silybin and N-acetylcysteine, the patient recovered being discharged 10 days post-ingestion with no organ complications. The prompt and appropriate therapy protocol for life-threatening amatoxins toxicity probably saved the patient's life as oral absorption was decreased and also supportive care was immediately started.

Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.

Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.

A Case Study: Rare Lepiota brunneoincarnata Poisoning.

Amatoxin poisoning from the genus Lepiota may have a deadly outcome, although this is not seen as often as it is from the genus Amanita. In this report, we present a patient who was poisoned by a sublethal dose of Lepiota brunneoincarnata mushrooms. The patient was hospitalized 12 hours after eating the mushrooms. The patient's transaminase levels increased dramatically starting on day 4. Aspartate transaminase peaked at 78 hours. Starting at 1265 IU/L, alanine transaminase peaked at 90 hours at 5124 IU/L. The patient was discharged on day 8 to outpatient care, and his transaminase levels returned to normal ranges in the subsequent days. A toxin analysis was carried out on the mushrooms that the patient claimed to have eaten. Using reversed-phase high-performance liquid chromatography analysis, an uptake of approximately 19.9 mg of amatoxin from nearly 30 g of mushrooms was calculated. This consisted of 10.59 mg of α-amanitin, 9.18 mg of ß-amanitin, and 0.16 mg of γ-amanitin. In conclusion, we present a patient from Turkey who was poisoned by L. brunneoincarnata mushrooms.

Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Evaluation and comparison of alpha- and beta-amanitin toxicity on MCF-7 cell line.

BACKGROUND/AIM: Alpha- and beta-amanitins are the main toxins of the poisonous Amanita phalloides mushroom. Although there are many studies available concerning alpha-amanitin, there are limited data about beta-amanitin in the literature. Therefore, this study is aimed at comparing the toxic effects of alpha- and beta-amanitin on the MCF-7 cell line. MATERIALS AND METHODS: The alpha- and beta-amanitins used for this research were purified from Amanita phalloides by preparative high-performance liquid chromatography. The MCF-7 breast cancer cell line was used, and specific concentrations of the toxins (100, 10, 1, 0.1, and 0.01 µg/mL) were applied to the cells. The MTT test was performed to determine the level of toxicity, and the quantity of protein in the cell was measured using the biuret test. RESULTS: The aLpha-amanitin showed a higher toxicity at 36 h, while the highest inhibition of protein synthesis by the beta-amanitin was observed at 24 h. CONCLUSION: It was shown that the beta-amanitin may be responsible for toxicity, like alpha-amanitin, in Amanita phalloides mushroom poisoning. The early inhibition of protein synthesis for beta-amanitin might be useful for future experiments and research.

Clinical importance of toxin concentration in Amanita verna mushroom.

Poisoning from Amanita group of mushrooms comprises approximately 3% of all poisonings in our country and their being responsible for nearly the entire fatal mushroom poisonings makes them important. These mushrooms contain primarily two types of toxins, amatoxins and phallotoxins. Phallotoxins have a more limited toxicity potential and they primarily consist of phalloidin (PHN) and phallacidin (PCN). Amatoxins, on the other hand, are very toxic and they primarily consist of alpha-amanitin (AA), beta-amanitin (BA) and gamma-amanitin (GA). Toxin levels can vary among various species, even among varieties of the same species, of Amanita mushroom family. Revealing the differences between the toxin compositions of the Amanita species that grow in our region may contribute to the clinics of poisonings. Our study aims at showing in detail the toxin levels in various parts of Amanita verna mushroom. A. verna mushrooms needed for toxin analysis were collected from Kozak Plateau near Ayvalik county of Balikesir, Turkey in April 2013. The mushrooms were divided into their parts as pileus, gills, stripe and volva. Following the procedures required before the analysis, the AA, BA, GA, PHN and PCN levels were measured using the RP-HPLC method. While the lowest level of amatoxin was in the volva of the mushroom, the highest was measured in the gills. This was followed by pileus and stripe where the levels were close to each other. Similarly, the highest level of phallotoxin was measured in the gills. Gamma toxin and phalloidin were at lower amounts than the other toxins. A. verna is frequently confused with edible mushrooms with white caps due to its macroscopic similarity. If just one of them is eaten by mistake by an adult person with no mushroom experience, it can easily poison them. The amount of amatoxin is more as compared to Amanita phalloides and A. phalloides var. alba. Particularly, the AA and BA levels are approximately three times higher, whereas GA levels are lower. Similarly, the level of PCN is approximately four times higher as compared to A. phalloides and A. phalloides var. alba; by contrast, the level of PNH is about a half of theirs. In summary, it can be said that A. verna is a more toxic mushroom than A. phalloides and has a higher rate of mortality. With our study, the amatoxin and phallotoxin concentrations and distribution in A. verna mushrooms were shown in detail for the first time and it would be useful to carry out more similar studies with other members of Amanita family growing in various parts of the world.