UCIBIO, REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.
UC-Biotech, CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3060-197, Cantanhede, Portugal.
Arch Toxicol. 2020 Jun;94(6):2061-2078. doi: 10.1007/s00204-020-02718-1. Epub 2020 Mar 19.
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.
α-鹅膏蕈碱在鹅膏蕈碱中毒中起着关键作用。肝脏是α-鹅膏蕈碱毒性的主要靶标，虽然 RNA 聚合酶 II（RNA Pol II）转录抑制是 α-鹅膏蕈碱毒性的公认机制，但其他可能的毒理学途径仍有待阐明。本研究旨在评估 α-鹅膏蕈碱在 HepG2 细胞中的肝毒性机制。还在该细胞模型和通透的 HeLa 细胞中测试了推测的解毒剂的潜在保护作用。当通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）还原和中性红摄取测定评估时，α-鹅膏蕈碱（0.1-20 μM）显示出时间和浓度依赖性细胞毒性。此外，α-鹅膏蕈碱以浓度和时间依赖的方式降低新生 RNA 的合成。虽然 α-鹅膏蕈碱没有诱导线粒体膜电位变化，但它导致细胞内 ATP 水平显著增加，而用寡霉素（一种 ATP 合酶抑制剂）孵育并不能防止这种增加。关于细胞氧化还原状态，α-鹅膏蕈碱没有增加活性物质的产生，但导致总谷胱甘肽和还原型谷胱甘肽的显著增加，而用γ-谷氨酰半胱氨酸合成酶抑制剂丁硫氨酸亚砜胺预先孵育则可以消除这种增加。在 HepG2 细胞中，测试的解毒剂[N-乙酰半胱氨酸、水飞蓟素、苄青霉素和多粘菌素 B（PolB）]没有一种能对抗 α-鹅膏蕈碱诱导的细胞毒性，也没有逆转毒素对通透的 HeLa 细胞中新生 RNA 的抑制作用。尽管如此，PolB 在高浓度下干扰 RNA Pol II 活性，但对观察到的 α-鹅膏蕈碱细胞毒性没有影响。本文描述了 α-鹅膏蕈碱的新肝毒性机制，但在临床上使用的解毒剂中观察到的缺乏保护作用可能反映了对其真正保护机制的了解不足，也可能解释了它们相对较低的临床疗效。
