Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.

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.

In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (α-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by α-AMA have not been fully characterized. In this study, we analyzed the secretome of α-AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1440 and 208 proteins were quantified in cell media and mouse serum, respectively. Based on the bioinformatics results for the commonly downregulated proteins in cell media and mouse serum, we identified complement component 3 (C3) as a marker for α-AMA-induced hepatotoxicity. Through western blot in cell secretome and C3 ELISA assays in mouse serum, we validated α-AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that α-AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of α-AMA-induced hepatotoxicity.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s43188-022-00163-z.

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

Mistakenly picking and eating poisonous mushrooms can cause acute poisoning. In August 2020, Qingdao Hospital of Traditional Chinese Medicine handled a poisonous mushroom poisoning incident, conducted epidemiological investigation on all poisoned patients, collected suspicious food, clinical manifestations, clinical test results and treatment conditions, and identified the mushrooms as Amanita fuliginea poisoning after morphological identification. In this incident, 6 people ate grey goose paste, of which 4 were sick with a incubation period of 6~12 h. The clinical manifestations were gastrointestinal symptoms such as nausea, vomiting and diarrhea, liver and kidney damage. After symptomatic support treatment, hemoperfusion or continuous hemofiltration treatment, the patients were cured and discharged. It is suggested to strengthen the popular science education on poisonous mushroom poisoning and improve the ability of identification and clinical treatment of poisonous mushrooms in grass-roots medical institutions.
误采、误食毒蘑菇可引起急性中毒，2020年8月青岛市中医医院处置一起毒蘑菇中毒事件，对所有中毒患者进行流行病学调查，收集可疑食物、临床表现、临床化验结果和治疗情况等资料，对蘑菇进行形态学鉴定后，鉴定为灰花纹鹅膏中毒。该起事件共有6人进食灰花纹鹅膏，其中4人发病，潜伏期6~12 h，临床表现为恶心、呕吐和腹泻等胃肠道症状，肝肾损伤，经过对症支持治疗、血液灌流或连续性血液滤过治疗等救治方案后，患者均治愈出院。建议加强针对毒蘑菇中毒的科普教育，提高基层医疗机构的毒蘑菇鉴别与临床救治能力。.

Omphalotus japonicus is a major toxic mushroom in Japan. When food poisoning caused by O. japonicus occurs, quick and accurate identification using a method that does not rely on morphological discrimination is required. Because the loop-mediated isothermal amplification (LAMP) method meets these requirements, we developed a LAMP method for detecting O. japonicus. Amplification occurred within 60 min, and the presence or absence of O. japonicus was confirmed within 2 h, including the DNA extraction protocol. The LAMP method did not show cross-reactivity with 13 species of edible mushrooms, had high specificity toward O. japonicus, and had sufficient detection sensitivity even in a mixed mushroom sample containing 1% O. japonicus. Additionally, O. japonicus could be detected in simulated food poisoning samples of heated and digested mushrooms, and in actual food poisoning residual samples.