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In vitro mechanistic studies on α-amanitin and its putative antidotes

  • Molecular Toxicology
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A Correction to this article was published on 17 April 2020

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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.

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  • 17 April 2020

    In the original publication of the article.

Abbreviations

BSA:

Bovine serum albumin

BSO:

Buthionine sulfoximine

BPNC:

Benzylpenicillin

DMEM:

Dulbecco’s modified eagle medium

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

EU:

5-Ethynyl uridine

EUTP:

5-Ethynyl uridine-triphosphate

HBSS:

Hanks’ balanced salt solution

MMP:

Mitochondrial membrane potential

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NAC:

N-Acetyl cysteine

NR:

Neutral red

SIL:

Silibinin

PBF:

Physiological buffer with Ficoll

PBS:

Phosphate buffer solution

PolB:

Polymyxin B

RNA Pol II:

RNA polymerase II

RS:

Reactive species

ROS:

Reactive oxygen species

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Acknowledgements

We want to acknowledge Professor Marteijn from Erasmus Medical Center, Rotterdam, Netherlands for the RPB1-GFP cell line. This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by national funds by the FCT within the project PTDC-DTP-FTO-4973-2014 – POCI-01-0145-FEDER 016545. This work was also supported by the Applied Molecular Biosciences Unit – UCIBIO through UID/MULTI/04378/2019 support with funding from FCT/MCTES through national funds. VMC acknowledges Fundação da Ciência e Tecnologia (FCT) for her grant (SFRH/BPD/110001/2015) that was funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/CP1334/CT0006.

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Authors and Affiliations

  1. UCIBIO, REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal

    Daniela Ferreira Rodrigues, Maria Lourdes Bastos, Vera M. Costa & Félix Carvalho

  2. UC-Biotech, CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3060-197, Cantanhede, Portugal

    Ricardo Pires das Neves & Alexandra T. P. Carvalho

  3. Institute for Interdisciplinary Research, University of Coimbra, 3030-789, Coimbra, Portugal

    Ricardo Pires das Neves & Alexandra T. P. Carvalho

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  1. Daniela Ferreira Rodrigues
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  2. Ricardo Pires das Neves
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  3. Alexandra T. P. Carvalho
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  4. Maria Lourdes Bastos
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  5. Vera M. Costa
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Correspondence to Vera M. Costa or Félix Carvalho.

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Rodrigues, D.F., Pires das Neves, R., Carvalho, A.T.P. et al. In vitro mechanistic studies on α-amanitin and its putative antidotes. Arch Toxicol 94, 2061–2078 (2020). https://doi.org/10.1007/s00204-020-02718-1

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