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AbstractObjectiveMethodsResultsDiscussionConclusionAmatoxin-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.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.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, β-amanitin, and γ-amanitin. All patients eventually recovered.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.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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

ObjectiveTo explore toxicity and urinary metabolites of α-amanitin in mice with a 21-day toxicological experiment. MethodsTwenty ICR male mice were randomly divided into a control (n = 5) and three median lethal dose (LD50) groups (5 in each group) with a single intraperitoneal injection of α-amanitin at doses of 0.25, 0.35, and 0.5 mg/kg·bw. Daily urine excretion of the mice was collected from 1st to 7th day after the injection for detection of toxins with liquid chromatography-mass spectrometry. Poisoning symptoms, body weight, and death of the mice were recorded during the 21-day experiment. By the end of the observation, all mice were sacrificed for determinations of liver/kidney weight and organ coefficient, blood biochemical indicators and pathological examination. ResultsUrinary α-amanitin was detected in all mice of dose groups for 4 days after the toxic treatment and the urinary excretion of α-amanitin in the 1st day accounted for 67.00 % – 98.35% of total urinary excretion within 7 days after the intoxication. Six deaths were observed in the mice of dose groups from 3rd to 10th day after the toxic treatment. Compared to those in the control mice, significantly decreased kidney weight (0.48 ± 0.05 vs. 0.59 ± 0.05 g) and coefficient (1.35 ± 0.11% vs. 1.49 ± 0.09%) but increased serum creatinine (18.25 ± 8.80 vs. 11.30 ± 7.60 µmol/L) and alkaline phosphatase (111.50 ± 32.30 vs. 78.00 ± 13.20 U/L) were detected in the surviving mice of dose groups at the end of experiment (P < 0.05 for all); kidney pathological changes of the surviving mice were also observed mainly in renal cortex, including renal tubular lesion and a large amount of protein cast. ConclusionIn mice, urinary excretion of α-amanitin could be detected in the early intoxication stage (1st – 4th day) and kidney injury is a main toxic effect in late stage of intoxication.

Objective: This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin (AMA) in mushroom. Methods: In this study, the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/NHS, and further coupled with different carrier proteins to prepare immunogens and coating antigen. Afterwards, Balb/c mice were immunized with the immunogen to prepare monoclonal antibodies. Based on the obtained antibody, an indirect competitive ELISA method for the detection of amanitin in mushroom was established by optimizing the working concentrations of coating antigen and antibody, coating conditions, blocking conditions, working concentrations of enzyme labeled secondary antibody and incubation time. Finally, the sensitivity, recovery rate, intra batch and inter batch variation of the established method were evaluated. Results: The molecular weight of the synthesized hapten in this study was 1033.12, and the coupling ratio of the immunogen identified by MALDI-TOF was about 10.03. Based on hybridoma technology, the IC50 of the mouse monoclonal antibody 13H4 was 1.91 μg/L. Based on the obtained monoclonal antibody, the detection limit of the established ic-ELISA method for amanitin in mushroom was 0.88 μg/kg, the recovery rate was 85.66%~113.05%, the intra-assay coefficient of variation was 5.35%~9.54%, and the inter-assay coefficient of variation was less than 15%. Conclusion: The ic-ELISA method established in this study had high accuracy, precision, sensitivity, and stable performance. It provided a simple, reliable and rapid detection method for the analysis of the toxicogen of sudden mushroom poisoning.

Silymarin is an herbal remedy, commonly called milk thistle, or St. Mary's Thistle, and has been used for over 2000 years. It has been available as a capsule of the plant extract in Europe since 1974 to treat hepatic disorders. To date toxicologists have relied on animal studies, human case series, or retrospective reviews to decide on its use. In the U.S. the ability to use IV silibinin, its pharmacologically active purified flavonolignan, is hindered by its lack of availability as a Food and Drug Administration approved pharmaceutical preparation. This commentary reviews the in vitro studies, animal studies, and human retrospective analyses which form the basis for its clinical use. Despite the numerous publications, summarized in this issue in a systematic review, the mortality rate from Amanita mushroom ingestion remains stubbornly the same over four decades of use, and hovers around 10%. Although in the retrospective systematic review the use of silibinin, or penicillin, compared to routine care is statistically significantly superior when the primary outcome is fatality. Despite this there is no quality randomized trial to definitively demonstrate its utility. While, intravenous silibinin has a low toxicity, unanswered is whether it is useful in protecting the liver in cases of amanitin-containing mushrooms toxicity, and whether earlier administration would likely improve outcomes. [ABSTRACT FROM AUTHOR]

Mushroom poisoning is the most important cause of death in food-borne poisoning in China, mainly caused by amanitin, which is caused by rapid progression, complex mechanism and latency. Early identification, diagnosis and treatment are important to improve the prognosis of fatal mushroom poisoning. This article analyzes the clinical characteristics, identification process and treatment of 14 patients with amanitin-containing Galerina sulciceps mushroom poisoning in a family, so as to improve the identification ability of the first physician in recognizing and managing early-stage mushroom poisoning, and to increase the cure rate through early bundle therapy of mushroom poisoning.

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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, β, 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. [Display omitted] • The cytotoxicity of amanitins differs greatly in various human cell lines. • The variable cytotoxicity is mainly due to different expression levels of OATP1B3. • Knockdown of OATP1B3 in HepG2 cells prevented α-amanitin induced cytotoxicity. • OATP1B3 may be a crucial therapeutic target against amanitin-induced organ failure. [ABSTRACT FROM AUTHOR]

Introduction: The consumption of mushrooms (Basidiomy-cota) in Poland is one of the highest in Europe. It is particularly high in the 3rd quarter of each year, which is accompanied by an increase in the number of mushroom poisonings. This study aims to present difficulties with microscopic identification, the most popular method for diagnosing mushroom poisoning in hospital settings, when it comes to detecting Amanita phalloides spores in biological material. Materials and methods: Spore analysis was carried out using aqueous solutions containing reference spores of different mushrooms: death cap (Amanita phalloides), parasol mushroom (Mac-rolepiota procera), field mushroom (Agaricus campestris), yellow knight mushroom (Tricholoma equestre), and green cracking russula (Russula virescens). The spore analysis was also carried out for a meal (soup) containing selected spores. Spores were identified using a light microscope and staining with Sudan III and Meltzer's reagent. A statistical analysis of mushroom poisoning cases was also performed at the Department of Clinical and Forensic Toxicology, Pomeranian Medical University in Szczecin using records for 2015-2019. Conlusions: Analysis of data from 2015-2019 from the Department of Clinical and Forensic Toxicology at the Pomeranian Medical University in Szczecin showed a marked increase in mushroom poisoning cases in the 3rd quarter of each year. Analysis of materials containing Amanita phalloides spores revealed their high similarity to oil drops and other cell structures present in biological material, resulting in the low reliability of microscopic identification. Therefore, as the absence of Amanita phalloides spores in the tested biological material does not rule out poisoning with this mushroom, a more advanced instrumental analysis (ELISA, LC/MS) is recommended. [ABSTRACT FROM AUTHOR]

The amanitins (namely α- and β-amanitin) contained in certain mushrooms are bicyclic octapeptides that, when ingested, are responsible for potentially lethal hepatotoxicity. M101 is an extracellular hemoglobin extracted from the marine worm Arenicola marina. It has intrinsic Cu/Zn-SOD-like activity and is currently used as an oxygen carrier in organ preservation solutions. Our present results suggest that M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development. [Display omitted] • α- and β-amanitin lead to cell death and mitochondrial reactive oxygen species production in differentiated HepaRG cells. • M101 is effective in reducing amanitin-induced cell death and mitochondrial reactive oxygen species production in vitro. • M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development. [ABSTRACT FROM AUTHOR]

Alpha‐amanitin, an extremely toxic bicyclic octapeptide extracted from the death‐cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody‐drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans‐hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp‐analogs to alter the conformational and H‐bonding properties of Hyp in amanitin. [ABSTRACT FROM AUTHOR]

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, β-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 β-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. [Display omitted] • This study found significant variations in amatoxin concentrations within same mushroom species and location. • This case study highlights the need for prompt treatment in wild mushroom poisoning, particularly when the incubation period exceeds 6 hours. • This case demonstrates the importance of appropriate treatment for the prognosis of the patient. • This case highlights the necessity of providing training to grassroots medical institution staff to ensure their understanding of common toxins. [ABSTRACT FROM AUTHOR]

Abstract Background Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway. Results The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the house-keeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. Conclusions To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.

Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms. [ABSTRACT FROM AUTHOR]

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), β-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. • There are limited data on the toxin content and profile of Galerina marginata. • Beta-amanitin levels were higher than alpha-amanitin in fungi collected from different regions of Turkey. • Gamma-amanitin levels were less than the detection limits. • It did not contain any phallotoxins. • For the first time, the toxin content of G. marginata was confirmed by mass spectrometry. [ABSTRACT FROM AUTHOR]

Amatoxins are deadly wild mushroom toxins that cause severe poisoning in humans, from diarrhea to organ dysfunction. Mortality can be as high as 80% if no specific treatment is applied. In this study, separation and determination methods were developed for the simultaneous determination of 7 toxins belonging to Amanita phalloides. After extraction and purification of Amanita phalloides (death cap) wild mushrooms, toxins were detected using HPLCESI MS and exact mass with time of flight MS (TOF-MS) in positive ionization mode. In addition, it was observed that the toxins of α- and β-amanitine could be differentiated from each other thanks to HR-MS detection in case of their close retention (Rt) in LC. The presence of two toxins at the same retention point in the chromatogram was detected by differentiating the molecular ion mass (920.3514 DA) of the α-amanitine with the HR-MS (920.3696 DA). By comparing both optimized methods with each other, faster and broad-spectrum toxin identification was achieved compared to existing methods. In this study, the rapid and accurate identification of seven toxins simultaneously with the same method is of great importance. It will make important contributions to the treatment process, especially by determining that poisoning is caused by wild mushrooms. [ABSTRACT FROM AUTHOR]

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 β-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.

Backgorund & Aims: Mushroom poisoning with Amanita phalloides or similar species can lead to liver failure with 10‐30% mortality rates. We aimed at defining the prognostic value of urinary amatoxin quantification in patients with hepatotoxic mushroom poisoning. Methods: Data from 32 patients with hepatotoxic mushroom poisoning (Hospital Clínic Barcelona, 2002‐16) in whom urinary amatoxins were determined (ELISA) were retrospectively reviewed. Correlations between urinary amatoxin and collected baseline variables with outcomes including hepatotoxicity (ALT>1000 U/L), severe acute liver injury (ALI, prothrombin <50%), acute liver failure (ALF, ALI and encephalopathy), transplantation/death and hospital length‐of‐stay, were evaluated. Results: 12/32 patients developed increased aminotransferase activity. Among the 13/32 amatoxin negative patients, 1 developed ALI and 12/13 no hepatotoxicity. Among the 19/32 amatoxin positive patients, 8/19 (42%) developed hepatotoxicity, including 5 who progressed to severe ALI, of whom 3 developed ALF (2 deaths, 1 transplantation). Urinary amatoxin and prothrombin were independent predictors of hepatotoxicity, ALT peak values (along with age) and hospital length‐of‐stay. In positive amatoxins patients, urinary concentrations > 55 ng/ml (or a baseline prothrombin ≤ 83%), were associated to hepatotoxicity (presented by 8/9 patients with ALT>1000 U/L). Among 5 patients with urinary amatoxin ≥ 70 ng/ml, 4 developed severe ALI. Conclusions: In patients with hepatotoxic mushroom poisoning, a negative urinary amatoxin quantification within 72h of intake ruled out the risk of hepatotoxicity in 92% of patients, whereas positive urinary amatoxins were associated with hepatotoxicity and severe ALI. Concentrations >55 ng/ml and ≥ 70 ng/ml were predictive of hepatotoxicity and severe ALI, respectively. See Editorial on Page 1016 [ABSTRACT FROM AUTHOR]

Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and β-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37–2890 pg/mL, and β-amanitin was found in seven plasma samples ranging from

Antibody–drug conjugates (ADCs) are an emerging class of anticancer therapeutics, delivering highly cytotoxic molecules directly to cancer cells. ADCs are composed of an antibody, a small molecule drug, and a linker attaching one to another. Antibodies are directed to a large variety of antigens overexpressed on tumor cells, tumor vasculature, or tumor-supporting stroma. After internalization, the ADC is transferred to lysosomes where the cytotoxic component is released, finally killing the target cell. All ADCs are administered via intravenous injection. Once in the circulation, linker stability in plasma is of high importance. In vivo studies in animals address the release of payload over time and typically measure total antibody, conjugated ADC, and free drug. ADC development is driven by ICH (International Council for Harmonisation) guidelines S6(R1) and S9. Dose-limiting toxicities of current ADCs are mainly associated with the payload and correlate well between clinical trials and nonclinical studies in rodents and nonrodents. This mini review is intended to provide general information about ADCs in oncology and shall assist the toxicologic pathologist in correctly interpreting morphological findings acquired in toxicity studies with this entity. [ABSTRACT FROM AUTHOR]

Abstract The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect α-, β-, and γ-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N 10 -α-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N 10 -α-amanitin internal standard, precision and accuracy of α-amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1–200 ng/mL. β- and γ-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0–200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled α-amanitin with the ability to detect and confirm human exposures to α-, β-, and γ-amanitin. Highlights • An LC-MS/MS method for the detection of α-, β-, and γ-amanitin was developed. • The development included an evaluation of an 15N 10 a-amanitin internal standard to compensate for matrix effects. • The method can accurately detect down α-, β-, and γ-amanitin to 1.0, 2.5, and 1.0 ng/mL in urine, respectively. • The 5N 10 α-amanitin internal standard significantly improved precision for α-amanitin. [ABSTRACT FROM AUTHOR]

It is believed that Lepiota cristata is inedible and slightly poisonous, so it should always be avoided. This study analyzed the alpha, beta and gamma amanitin, phalloidin (PHN) and phallacidin (PCN) content of L. cristata collected from different regions of Turkey. Toxin levels have been assessed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with ultraviolet detectors. In this study, the L. cristata mushroom was not found to contain any amatoxins or phallotoxins. However, this finding does not mean that this mushroom is edible. New studies regarding the toxin content of L. cristata may be useful to understand the toxicity of this species. [ABSTRACT FROM AUTHOR]

Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled to UV absorbance and mass spectrometry, we analyzed the spectrum of peptide toxins present in six Italian species of Lepiota, including multiple samples of three of them collected in different locations. Field taxonomic identifications were confirmed by sequencing of the internal transcribed spacer (ITS) regions. For comparison, we also analyzed specimens of Amanita phalloides from Italy and California, a specimen of A. virosa from Italy, and a laboratory-grown sample of Galerina marginata. α-Amanitin, β-amanitin, amanin, and amaninamide were detected in all samples of L. brunneoincarnata, and α-amanitin and γ-amanitin were detected in all samples of L. josserandii. Phallotoxins were not detected in either species. No amatoxins or phallotoxins were detected in L. clypeolaria, L. cristata, L. echinacea, or L. magnispora. The Italian and California isolates of A. phalloides had similar profiles of amatoxins and phallotoxins, although the California isolate contained more β-amanitin relative to α-amanitin. Amaninamide was detected only in A. virosa.

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. [ABSTRACT FROM AUTHOR]

Background/aim: This study aimed to evaluate oxidative stress markers of liver tissue in a mouse a-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 a-amanitin-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

Background: The cyclic peptide toxins of Amanita mushrooms, such as α-amanitin and phalloidin, are encoded by the "MSDIN" gene family and ribosomally biosynthesized. Based on partial genome sequence and PCR analysis, some members of the MSDIN family were previously identified in Amanita bisporigera, and several other members are known from other species of Amanita. However, the complete complement in any one species, and hence the genetic capacity for these fungi to make cyclic peptides, remains unknown. Results: Draft genome sequences of two cyclic peptide-producing mushrooms, the "Death Cap" A. phalloides and the "Destroying Angel" A. bisporigera, were obtained. Each species has ~30 MSDIN genes, most of which are predicted to encode unknown cyclic peptides. Some MSDIN genes were duplicated in one or the other species, but only three were common to both species. A gene encoding cycloamanide B, a previously described nontoxic cyclic heptapeptide, was also present in A. phalloides, but genes for antamanide and cycloamanides A, C, and D were not. In A. bisporigera, RNA expression was observed for 20 of the MSDIN family members. Based on their predicted sequences, novel cyclic peptides were searched for by LC/MS/MS in extracts of A. phalloides. The presence of two cyclic peptides, named cycloamanides E and F with structures cyclo(SFFFPVP) and cyclo(IVGILGLP), was thereby demonstrated. Of the MSDIN genes reported earlier from another specimen of A. bisporigera, 9 of 14 were not found in the current genome assembly. Differences between previous and current results for the complement of MSDIN genes and cyclic peptides in the two fungi probably represents natural variation among geographically dispersed isolates of A. phalloides and among the members of the poorly defined A. bisporigera species complex. Both A. phalloides and A. bisporigera contain two prolyl oligopeptidase genes, one of which (POPB) is probably dedicated to cyclic peptide biosynthesis as it is in Galerina marginata. Conclusion: The MSDIN gene family has expanded and diverged rapidly in Amanita section Phalloideae. Together, A. bisporigera and A. phalloides are predicted to have the capacity to make more than 50 cyclic hexa-, hepta-, octa-, nona- and decapeptides. [ABSTRACT FROM AUTHOR]

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.

The authors describe two kinds of rapid assays for the determination of amatoxins in mushrooms. The first is an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase. The second is a rapid immunochromatographic assay that uses colloidal gold as a red label (CG-ICA). Both are based on the use of a well-characterized recombinant single chain variable fragment antibody (named scFv-A4). The half-maximum inhibition concentrations (IC50) of α-amanitin, β-amanitin and γ-amanitin are 78, 85 and 90 ng⋅mL‾, and the limits of detection (LODs; for IC15) are 1.9, 2.1 and 2.8 ng⋅mL‾. The method was applied to the determination of amanitins in mushrooms, and the LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were found to be 4.9, 6.4 and 8.3 ng⋅mL‾. The visual minimum detection limits of the optimized CGIA are 4 and 6 ng⋅mL‾ for mushroom samples. The test can be performed within 10 min. The results of the analysis of spiked samples showed that the CG-IA can rapidly and semi-quantitatively quantify amatoxins in mushroom samples on site and at low costs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate beneficial effects of erdosteine in the alpha amanitine-induced hepatotoxicity in mice. Three hours after giving alpha amanitin (0.5 mg/kg, i.p.) to the mice, they were administered silibinin (50 mg/kg/d, i.p.) or erdosteine (100 mg/kg/d, oral) therapies once a day for 3 d. A histopathological examination of their liver tissues was carried out 24 h after the last treatment; transaminase levels, blood urea nitrogen, urea, and creatinine were analyzed in serum. Erdosteine showed a beneficial effect by significantly improving the functional parameters particularly in alpha amanitin-induced hepatotoxicity and partially in renal toxicity. In the histopathological evaluation, the toxicity that was generated with alpha amanitin was significantly reduced by erdosteine, showing a possible hepatoprotective effect. [ABSTRACT FROM AUTHOR]