Abstract

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.

中文翻译：

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α-amanitin及其推定解毒剂的体外机理研究。

摘要

α-甘露素在伞形毒蕈中起关键作用中毒。肝脏是α-amanitin毒性的主要靶标，而RNA聚合酶II（RNA Pol II）转录抑制是α-amanitin毒性的公认机制，但其他可能的毒理学途径尚待阐明。这项研究旨在评估HepG2细胞中α-amanitin肝毒性的机制。在该细胞模型和透化的HeLa细胞中也测试了假定解毒剂的假定保护作用。当通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑（MTT）还原和中性红进行评估时，α-天冬氨酸（0.1–20 µM）显示出时间和浓度依赖性的细胞毒性。摄取测定。此外，α-amanitin以浓度和时间依赖性方式减少新生RNA的合成。虽然α-amanitin不会诱导线粒体膜电位发生变化，但会引起细胞内ATP水平的显着增加，但与ATP合成酶抑制剂寡霉素孵育并不能阻止这种现象。关于细胞的氧化还原状态，α-amanitin不会增加反应物种的产生，但会导致谷胱甘肽的总量增加和减少，这是通过与γ-谷氨酰半胱氨酸合酶抑制剂丁硫氨酸亚砜亚胺预孵育而取消的。没有经过测试的解毒剂[ 通过与γ-谷氨酰半胱氨酸合酶抑制剂丁硫氨酸亚砜肟一起预孵育而被取消。没有经过测试的解毒剂[ 通过与γ-谷氨酰半胱氨酸合酶抑制剂丁硫氨酸亚砜肟一起预孵育而被取消。没有经过测试的解毒剂[N-乙酰半胱氨酸，水飞蓟宾，苄青霉素和多粘菌素B（PolB）]赋予了任何保护作用，可阻止HepG2细胞中α-amanitin诱导的细胞毒性或逆转通透性HeLa细胞中由毒素引起的新生RNA的抑制作用。尽管如此，PolB在高浓度下仍会干扰RNA Pol II活性，尽管不会影响α-amanitin所观察到的细胞毒性。本文描述了α-amanitin的新肝毒性机制，但在临床使用的解毒剂中观察到的保护作用不足可能反映出对其真正保护机制的认识不足，并可能解释了其相对较低的临床疗效。