Amanita phalloides is an extremely toxic and frequently lethal mushroom, especially due to its high levels of powerful toxins such as α-amanitin, a well-established RNA polymerase II inhibitor. α-Amanitin intoxications have been associated with acute kidney injury and renal failure, besides its well-known hepatotoxic effects. Currently, no effective antidote against α-amanitin toxicity exists. Recent in vivo studies have shown that polymyxin B (PolB) decreases α-amanitin toxicity and that the associated renal damage is largely decreased by that antibiotic. Thus, this work aimed to characterize α-amanitin cytotoxicity in HK-2 cells and evaluate PolB’s putative antidotal effectiveness in this in vitro system.
HK-2 cells were exposed to α-amanitin (0.01-10 µM) for 24- or 48h. Additionally, 1 and 2h incubations with α-amanitin (1-10 µM) followed by a 48-h α-amanitin-free period were also performed to assess the effect of early entrance of the toxin to the cells. Cytotoxicity was evaluated by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. To assess PolB putative protective effects, two paradigms were used: (i) 30 min pre-incubation with PolB followed by 48h incubation with α-amanitin (0.5 and 1 µM) or (ii) PolB co-incubation with α-amanitin (5 and10 µM) for 2h followed by a 48h drug/toxin-free period.
α-Amanitin toxicity was observed at 24h at concentrations above 1 µM in the MTT reduction assay. At 48h, α-amanitin 0.5 µM also caused significant cytotoxicity showing that the toxicity observed after the toxin was time- and concentration-dependent. Nonetheless, α-amanitin caused lower toxicity after shorter incubation periods (1 or 2h), possibly indicating a slow α-amanitin uptake by HK-2 cells. In fact, a 5 times higher concentration was needed to obtain a similar effect to the one obtained under a 48h continuous incubation period. In addition, PolB did not confer protection any against α-amanitin cytotoxicity in either the long exposure or the low exposure α-amanitin approaches tested.
To sum up, α-amanitin led to cytotoxicity effects on kidney cells at clinical relevant concentrations. The effectiveness of a previously described antidote, PolB, was not verified in vitro, which highlights the importance of further investigation on this antidotal strategy and its mechanisms.
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. 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.