Toxicokinetics of α- and ß-amanitin in mice following single and combined administrations: Simulating in vivo amatoxins processes in clinical cases.

α-Amanitin and ß-amanitin, two of the most toxic amatoxin compounds, typically coexist in the majority of Amanita mushrooms. The aim of this study was to use a newly developed ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method to determine the toxicokinetics and tissue distribution of α- and ß-amanitin following single or combined oral (po) administration in mice. α-Amanitin and ß-amanitin administered at 2 or 10 mg/kg doses showed similar toxicokinetic profiles, except for peak concentration (Cmax). The elimination half-life (t1/2) values of α-amanitin and ß-amanitin in mice were 2.4-2.8 h and 2.5-2.7 h, respectively. Both α- and ß-amanitin were rapidly absorbed into the body, with times to reach peak concentration (Tmax) between 1.0 and 1.5 h. Following single oral administration at 10 mg/kg, the Cmax was significantly lower for α-amanitin (91.1 µg/L) than for ß-amanitin (143.1 µg/L) (p < 0.05). The toxicokinetic parameters of α-amanitin, such as t1/2, mean residence time (MRT), and volume of distribution (Vz/F) and of ß-amanitin, such as Vz/F, were significantly different (p < 0.05) when combined administration was compared to single administration. Tissues collected at 24 h after po administration revealed decreasing tissue distributions for α- and ß-amanitin of intestine > stomach > kidney > lung > spleen > liver > heart. The substantial distribution of toxins in the kidney corresponds to the known target organs of amatoxin poisoning. The content in the stomach, liver, and kidney was significantly higher for of ß-amanitin than for α-amanitin at 24 h following oral administration of a 10 mg/kg dose. No significant difference was detected in the tissue distribution of either amatoxin following single or combined administration. After po administration, both amatoxins were primarily excreted through the feces. Our data suggest the possibility of differences in the toxicokinetics in patients poisoned by mushrooms containing both α- and ß-amanitin than containing a single amatoxin. Continuous monitoring of toxin concentrations in patients' blood and urine samples is necessary in clinical practice.

Toxic Effects of Amanitins: Repurposing Toxicities toward New Therapeutics.

The consumption of mushrooms has become increasingly popular, partly due to their nutritional and medicinal properties. This has increased the risk of confusion during picking, and thus of intoxication. In France, about 1300 cases of intoxication are observed each year, with deaths being mostly attributed to Amanita phalloides poisoning. Among amatoxins, α- and ß-amanitins are the most widely studied toxins. Hepatotoxicity is the hallmark of these compounds, leading to hepatocellular failure within three days of ingestion. The toxic mechanisms of action mainly include RNA polymerase II inhibition and oxidative stress generation, leading to hepatic cell apoptosis or necrosis depending on the doses ingested. Currently, there is no international consensus concerning Amanita phalloides poisoning management. However, antidotes with antioxidant properties remain the most effective therapeutics to date suggesting the predominant role of oxidative stress in the pathophysiology. The partially elucidated mechanisms of action may reveal a suitable target for the development of an antidote. The aim of this review is to present an overview of the knowledge on amanitins, including the latest advances that could allow the proposal of new innovative and effective therapeutics.

Legalon® SIL: the antidote of choice in patients with acute hepatotoxicity from amatoxin poisoning.

More than 90% of all fatal mushroom poisonings worldwide are due to amatoxin containing species that grow abundantly in Europe, South Asia, and the Indian subcontinent. Many cases have also been reported in North America. Initial symptoms of abdominal cramps, vomiting, and a severe cholera-like diarrhea generally do not manifest until at least six to eight hours following ingestion and can be followed by renal and hepatic failure. Outcomes range from complete recovery to fulminant organ failure and death which can sometimes be averted by liver transplant. There are no controlled clinical studies available due to ethical reasons, but uncontrolled trials and case reports describe successful treatment with intravenous silibinin (Legalon® SIL). In nearly 1,500 documented cases, the overall mortality in patients treated with Legalon® SIL is less than 10% in comparison to more than 20% when using penicillin or a combination of silibinin and penicillin. Silibinin, a proven antioxidative and anti-inflammatory acting flavonolignan isolated from milk thistle extracts, has been shown to interact with specific hepatic transport proteins blocking cellular amatoxin re-uptake and thus interrupting enterohepatic circulation of the toxin. The addition of intravenous silibinin to aggressive intravenous fluid management serves to arrest and allow reversal of the manifestation of fulminant hepatic failure, even in severely poisoned patients. These findings together with the available clinical experience justify the use of silibinin as Legalon® SIL in Amanita poisoning cases.

The enterohepatic circulation of amanitin: kinetics and therapeutical implications.

BACKGROUND: Amatoxin poisoning induces a delayed onset of acute liver failure which might be explained by the prolonged persistence of the toxin in the enterohepatic circulation. Aim of the study was to demonstrate amanitin kinetics in the enterohepatic circulation. METHODS: Four pigs underwent α-amanitin intoxication receiving 0.35 mg/kg (n=2) or 0.15 mg/kg (n=2) intraportally. All pigs remained under general anesthesia throughout the observation period of 72 h. Laboratory values and amanitin concentration in systemic and portal plasma, bile and urine samples were measured. RESULTS: Amanitin concentrations measured 5h after intoxication of 219±5ng/mL (0.35 mg/kg) and 64±3 (0.15 mg/kg) in systemic plasma and 201±8ng/mL, 80±13ng/mL in portal plasma declined to baseline levels within 24h. Bile concentrations simultaneously recorded showed 153±28ng/mL and 99±58ng/mL and decreased slightly delayed to baseline within 32 h. No difference between portal and systemic amanitin concentration was detected after 24h. CONCLUSIONS: Amanitin disappeared almost completely from systemic and enterohepatic circulation within 24 h. Systemic detoxification and/or interrupting the enterohepatic circulation at a later date might be poorly effective.

The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin alpha-amanitin.

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.

Cap mushroom poisonings.

This paper presents species of fungi of high toxicity. Their consumption might have serious consequences for health and in many cases it might lead to death. Toxic compounds present in fungi have also been characterised, mechanisms of their toxic activity have been presented and clinical symptoms of poisoning have been described. Hallucinogenic mushrooms have also been mentioned as they have recently become a serious problem: many people use them to intoxicate themselves. There are also species of mushrooms that can be consumed under certain conditions since they can occasionally trigger off serious disturbances for the functioning of organisms.

Studies on the possible mechanisms of protective activity against alpha-amanitin poisoning by aucubin.

Aucubin, an iridoid glucoside, was investigated to determine whether it has a stimulating effect on alpha-amanitin excretion in alpha-amanitin intoxicated rats, and whether there is binding activity to calf thymus DNA. High-performance liquid chromatography (HPLC) analysis of alpha-amanitin in rat urine allowed quantitative measurement of the alpha-amanitin concentration with a detection limit of 50 ng/ml. In this system, a group treated with both alpha-amanitin and aucubin showed that alpha-amanitin was excreted about 1.4 times faster than in the alpha-amanitin only treated group. Our previous results showed that the toxicity of alpha-amanitin is due to specific inhibition of RNA polymerase activity and the resultant blockage of the synthesis of certain RNA species in the nucleus. However, no significant activity change on RNA polymerase from Hep G2 cells was observed when aucubin was treated with alpha-amanitin at any concentration tested. Nevertheless, aucubigenin inhibited both DNA polymerase (IC50, 80.5 microg/ml) and RNA polymerase (IC50, 135.0 microg/ml) from the Hep G2 cells. The potential of both alpha-amanitin and aucubin to interact with DNA were examined by spectrophotometric analysis. Alpha-Amanitin showed no significant binding capacity to calf thymus DNA, but aucubin was found to interact with DNA, and the apparent binding constant (Kapp) and apparent number of binding sites per DNA phosphate (Bapp) were 0.45 x 10(4) M(-1) and 1.25, respectively.

Neural induction suppresses early expression of the inward-rectifier K+ channel in the ascidian blastomere.

1. Early expression of ion channels following neural induction was examined in isolated, cleavage-arrested blastomeres from the ascidian embryo using a two-electrode voltage clamp. Currents were recorded from the isolated, cleavage-arrested blastomere, a4-2, after treatment with serine protease, subtilisin, which induces neural differentiation as consistently as cell contact. 2. The inward-rectifier K+ current increased at the late gastrula stage shortly after the sensitive period for neural induction both in the induced (protease-treated) and uninduced cells. Ca2+ channels, characteristic of epidermal-type differentiation, and delayed-rectifier K+ channels and differentiated-type Na+ channels, characteristic of neural-type differentiation appeared much later than the inward-rectifier K+ channels, at a time corresponding to the tail bud stage of the intact embryo. 3. When cells were treated with subtilisin during the critical period for neural induction, the increase in the inward-rectifier K+ current from the late gastrula stage to the neurula stage was about three times smaller (3.67 +/- 1.74 nA, mean +/- S.D., n = 14) than in untreated cells (11.25 +/- 3.10 nA, n = 26). The same changes in the inward-rectifier K+ channel were also observed in a4 2 blastomeres which were induced by cell contact with an A4-1 blastomere. However, when cells were treated with subtilisin after the critical period for neural induction, the amplitude of the inward-rectifier K+ current was the same as in untreated cells. Thus the expressed level of the inward-rectifier K+ channel was linked to the determination of neural or epidermal cell types. 4. There was no significant difference in the input capacitance of induced and uninduced cells, indicating that the difference in the amplitude of the inward-rectifier K+ currents derived from a difference in the channel density rather than a difference in cell surface area. 5. The expression of the inward-rectifier K+ channel at the late gastrula stage was sensitive to alpha-amanitin, a highly specific transcription inhibitor. In both induced and uninduced cells, injection of alpha-amanitin at the 32-cell stage reduced the current density of the inward-rectifier K+ channel to about 2 nA/nF, corresponding to 13% of that recorded from uninjected cells. By contrast, the expression of the fast-inactivating-type Na+ current, which transiently increased along with the inward-rectifier K+ channel, was resistant to alpha-amanitin injection. 6. The dose of alpha-amanitin injected was controlled by monitoring co-injected fluorescent dye, fura-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of amatoxins in human poisoning: therapeutic implications.

The kinetics of alpha and beta amanitin were studied in 45 patients intoxicated with Amanita Phalloides. The amatoxins were analyzed by high performance liquid chromatography in plasma (43 cases), urine (35 cases), gastroduodenal fluid (12 cases), feces (12 cases) and tissues (4 cases). All patients had gastrointestinal symptoms and 43 developed an acute hepatitis. Two patients underwent successful liver transplantation. Eight patients, of whom three were children, died. The detection of amatoxins in the biological fluids was time dependent. The first sample was obtained at an average of 37.9 h post ingestion in the patients with positive results and at 70.6 h in the samples without detectable amatoxins. Plasma amatoxins were detected in 11 cases at 8 to 190 ng/mL for alpha and between 23.5 to 162 ng/mL for beta. In 23 cases amatoxins were detected in urine with a mean excretion per hour of 32.18 micrograms for alpha and 80.15 micrograms for beta. In 10 patients the total amounts eliminated in the feces (time variable) ranged between 8.4 and 152 micrograms for alpha amanitin and between 4.2 and 6270 micrograms for beta amanitin. In three of four cases amatoxins were still present in the liver and the kidney after day 5. Amatoxins were usually detectable in plasma before 36 h but were present in the urine until day 4. The rapid clearance indicates that enhanced elimination of amatoxins requires early treatment. Clearance of circulating amatoxins by day 4 spares the transplanted liver.

Hepatotoxic mushroom poisoning: diagnosis and management.

Hepatotoxic mushroom poisoning (due to Amanita, Lepiota and Galerina species) may be considered as a real medical emergency, since an early diagnosis and immediate treatment are required for a successful outcome. In this review the physio-pathological features and the clinical picture of amatoxin poisonings are described as the basis for diagnosis and therapeutic decisions. The treatment schedule proposed is analyzed in some points: Symptomatic and supportive measures, toxin removal and extraction procedures, and the possibility of using antidotes. Some parameters with prognostic significance are commented on. Finally, the mortality rate and its evolution throughout the present century is also considered.

Properties of phallotoxin uptake by basolateral plasma membrane vesicles from rat liver: evidence for a carrier-mediated transport.

The mechanism and driving forces for hepatocellular phallotoxin uptake were studied by a rapid-filtration technique using basolateral liver plasma membrane vesicles (blLPM). An inwardly directed Na+ gradient but not K+-gradient transiently stimulated taurocholate uptake into blLPM 1.4-1.7-fold above the equilibrium value (overshoot), demonstrating functionally intact vesicles. In contrast, overshooting phallotoxin uptake (1.15-1.2-fold intravesicular accumulation above equilibrium value) was observed in the presence of a K+ as well as of a Na+ gradient. Na+ could be replaced by K+ or Li+. In the presence of choline a distinct uptake reduction of 57% was seen. Counter-transport phenomena suggest phallotoxin transport rather than binding. Phallotoxin uptake was inhibited significantly by taurocholate, iodipamide and antamanide, but only slightly by alpha-amanitin. Creation of a negative intravesicular potential by altered accompanying anions or by valinomycin-induced K+ diffusion potential enhanced the initial uptake rate for phallotoxin, demonstrating rheogenic solute uptake. These findings provide evidence that hepatocellular uptake of phallotoxin is due to carrier-mediated transport. Hepatic uptake of phallotoxin is assumed to be driven by both a monovalent cation gradient (Na+ or K+) and a transmembranal potential difference.