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Abstract
Mushroom poisoning has emerged as a serious health problem. Among poisonous mushrooms, species containing cyclopeptides, such as Amanita phalloides, are the most toxic. Amatoxins and specially α-amanitin are responsible for the major deleterious effects. The liver and the kidney are the main targets for α-amanitin cytotoxicity, but hepatocellular effects represent the most lethal and the least treatable manifestation of amatoxin toxicity. Furthermore, there are no worldwide accepted guidelines for the treatment of amatoxinsintoxicated patients and none of the procedures or antidotes used so far has been clearly proven to have great clinical efficiency. Consequently, lethality is still high among intoxications with mushroom containing amatoxins.
The main aims of this dissertation were to characterize α-amanitin cytotoxicity in two human hepatic cell lines (HepG2 and HepaRG) and to evaluate the effect of several putative pharmacological active molecules towards α-amanitin toxicity, since the establishment of an accurate cell model for the evaluation of new antidotes for α-amanitin poisoning is critical in this field. Cytotoxicity of α-amanitin (0.01 – 20µM) in high and low density HepG2 and confluent and differentiated HepaRG cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays, following a 24 or 48- h incubation period. Additionally, the morphological changes induced by α-amanitin in high and low density HepG2 and in confluent and differentiated HepaRG cells were also assessed by phase contrast microscopy following a 48-h incubation period. The characterization of αamanitin cytotoxicity in high density HepG2 cells was complemented by assessing: 1) the production of reactive species with the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe; 2) total glutathione levels; 3) mitochondrial membrane potential; 4) ATP levels and 5) the effect cycloheximide (CHX), a protein synthesis inhibitor, and the effect of buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, on α-amanitin cytotoxicity following a 48-h incubation. Thereafter, the effect of previously described antidotes used in α-amanitin intoxications, namely N-acetylcysteine (NAC), silibinin (SIL) and benzylpenicillin (BPNC) and new putative antidotes polymyxin B (Pol) and cyclosporine A (CsA) was assessed towards the toxic effects of α-amanitin in both high and low density HepG2 and confluent and differentiated HepaRG cells.
Evaluation of the cytotoxic effects in HepG2 and HepaRG cells revealed that following a 48-h incubation, α-amanitin was more cytotoxic in differentiated HepaRG cells, which can be accounted to the presence and functional activity of the Na+-taurocholate cotransporting polypeptide (NTCP) uptake transporter. Concerning the characterization of α-amanitin cytotoxicity in high density HepG2 cells, this amatoxin (2 or 5µM) did not induce changes in mitochondrial membrane potential but α-amanitin 5µM caused a significant increase in intracellular ATP levels. CHX had no effect on the cytotoxicity caused by α-amanitin 5µM following a 48-h incubation. Regarding the cell redox status, α-amanitin (1, 2 or 5µM) did not induce changes in reactive species production but α-amanitin 2 and 5µM caused a tendency to increase total glutathione levels.
