A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography-mass spectrometry applied to Amanita toxins in food.

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and ß-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

[Phalloidin syndrome: role of Elisa-based assay for the detection of alpha- and gamma-amanitins in urine. Preliminary results]. / Syndrome phalloïdien: quelle est la place du dosage des alpha- et gamma-amanitines par ELISA (Bühlmann)? Résultats préliminaires.

UNLABELLED: After consumption of mushrooms containing amatoxins (Amanita, Lepiota, and Galerina species), symptoms usually develop after a long delay (>6 h). Initial symptoms start as severe gastroenteritis, progressing to liver failure and possibly death as a result of hepatic coma. Since the survival rate of poisoned patients is claimed to depend on the time of beginning of efficient treatment, fast and reliable assays for amatoxins in biological fluids are essential. Described analytical methods for amatoxins include high performance liquid chromatography and radioimmunoassay (RIA). Recently, a new enzyme-linked immunosorbent assay (Bühlmann Amanitin ELISA kit) has been introduced as an alternative method to RIA. This ELISA-based assay offers several advantages: no complex extraction procedure is required (vs. HPLC) and no safety precautions concerning radioactivity have to be taken (vs. RIA). From August 2004 to October 2005, a pilot study was performed to test the practicability and the clinical utility of this method in emergency situations. RESULTS: ten urines, 9 serums and 1 faeces from 10 patients suffering from acute gastroenteritis after mushroom ingestions (7 contaminated meals) were analyzed. Definitive diagnosis of amatoxin poisoning was made in 4 cases (3 contaminated meals) on the basis of the anamnesis, laboratory results, and clinical course. A patient developed a severe amatoxin poisoning with urinary amanitins level < 1.5 microg/L (urines were collected more than 72 h after mushroom ingestion). Two patients were paucisymptomatic with urinary amanitins levels >10 microg/L (urines were collected before the 36th hour). CONCLUSION: Urine is the sample of choice for the determination of amatoxins. The most critical factor to invalidate the usefulness of this analysis is time. After 36 h, the sensitivity is unreliable.

The toxicology of Amanita virosa: the destroying angel.

This paper examines the biology and medical consequences of ingesting the potentially lethal poisonous mushroom, Amanita virosa, the Destroying Angel. The fungus, its structure, distribution and toxic components are described. Symptoms of human poisoning by A. virosa are described, following the order of Homeopathic Repertories. Laboratory values for comparison with normal values of haematology, biochemistry and urine analyses are given.

Prophylaxis of cyanobacterial and mushroom cyclic peptide toxins.

The pathogenicity of virulent cyclic peptide toxins of the cyanobacterium, Microcystis aeruginosa, and the mushroom, Amanita phalloides, was prevented in mice by pretreatment with a variety of chemically unrelated agents including hydrocortisone, shellac, certain diazo and triazine dyes and cyclosporine. A. Despite the diverse nature of the protective agents, a feature commonly associated with protection was the ability to impair hepatic uptake of 51Cr-labeled sheep erythrocytes, a function of hepatic macrophages (Kupffer cells). In addition, several of the protective agents are known to affect other aspects of reticuloendothelial cell function. Therefore it seems likely that the hepatic macrophage is involved in the observed protection, although by what mechanism(s) is unknown. The most remarkable prophylaxis was seen with a single injection of Trypan red, which provided nonimmunologic protection against a lethal dose of a cyanobacterial toxin, cyanoginosin-LR, for periods up to 3 months.

[Cholinesterase activity as a prognostic test in phalloidine mushroom poisoning]. / Kholinesteraznata aktivnost kato prognostichen test pri faloidna gubna intoksikatsiia.

The phalloidine mushroom poisoning is an exogenic intoxication with specific action of the mushroom toxins on the liver and its functions. The dynamic follow up of serum cholinesterase activity in patients with phalloidine intoxication revealed that it was significantly decreased in the patients who died in comparison to those who survived. In the patients who survived and recovered the serum cholinesterase was moderately decreased and later increased. As a protein product synthesized in the liver the serum cholinesterase is a good marker of the protein-synthesizing function of the liver in patients with phalloidine intoxication and may be used as a prognostic test for its outcome.

[Functional disorders and structural changes in the liver in Amanita phalloides poisoning]. / Funktsionalni smushteniia i strukturni izmeneniia v cherniia drob pri faloidna gubna intoksikatsiia.

The liver functional disorders and structural changes were studied in 17 patients (children 6, adults 11, 4 to 70 years of age) with phalloidine intoxication. The clinical course of this severe and lethal exogenic intoxication, its characteristic clinical and laboratory features are discussed in relation to the morphological changes of the liver. The structural changes are similar in all patients independently of their age and the time of death. They are presented by fatty degeneration, acute toxic liver dystrophy and centrilobular necroses. These changes are produced by the specific action of the phalloidine toxins on the liver.

Acute phalloidin poisoning in dogs.

The acute toxic effects of phalloidin, a toxin from the green deathcup, Amanita phalloides, were tested in dogs. No fatalities occurred following intravenous injection; however, the biochemical parameters GPT, GOT, alkaline phosphatase, and total bilirubin yielded pathological values. Histologically the liver parenchyma revealed hemorrhagic necrosis and peliosis-like changes with penetration of red blood cells into hepatocytes.

New aspects of the origin of hepatocellular vacuoles. An ultrastructural study on rat and mice liver after different intoxications.

The stages of vacuolar formations in liver parenchymal cells of rats and mice following application of phalloidine, amanitine, o-phenylphenol, hexachlorophene and p-chloro-m-cresol were demonstrated by electron microscopic investigations. After treatment the intercellular spaces are markedly widened and in restricted regions exhibit large sacculi penetrating into the cytoplasm of adjacent hepatocytes. In cross-sections these invaginations frequently appear as intracellular vacuoles. Our present investigations, however, clearly demonstrate that these vacuoles are still in direct connection with the intercellular space. The vacuolar formation is more pronounced in older animals and it is suggested that an increased patho-physiological portal hypertension leads to the swelling of the intercellular space.

Decreased phalloidin response, phallotoxin uptake and bile acid transport in hepatocytes prepared from wistar rats treated chronically with diethylnitrosamine.

Isolated hepatocytes prepared from rats pretreated with diethylnitrosamine (0.5 mg/kg DENA/DAY P.O.) are less sensitive to phalloidin poisoning. They take up lower amounts of both phallotoxins and bile acids than controls. The degree of inhibition depends on the period of pretreatment.

[Amanita phalloides poisoning in Austria (author's transl)]. / Knollenblätterpilzvergiftungen in Osterreich.

An analysis of 28 cases of amanita phalloides poisoning serves as basis for a discussion of the clinical features and therapeutic problems involved. A critical review of recent experimental investigations in animals points to new possibilities in the treatment of amanita phalloides poisoning.

[The effects of flavonoids on rat liver during chronic palloidin intoxication/Morphological and biochemical studies (author's transl)]. / Zur Wirkung von Flavonoiden an der Rattenleber bei chronischer Phalloidin-Vergiftung. Morphologische und klinisch-chemische Untersuchungen.

Continuous intraperitoneal administration of phalloidin (0.5 mg/kg body weight/day) leads to an alteration of intracellular contractile acto-myosinfilaments in rat liver. The hepatocytes show an accumulation of fibrillar material with some loss of contractile function of the pericanalicular web. Biochemically an increase of serum transaminases and alkaline phosphatase occurs. Histochemically the liver exhibitis changes in the distribution of some hepatocellular enzymes. The influence of the flavonoid (+)-cyanidanol-3 on these phalloidin-induced lesions was studied by histochemical, immunofluorescence and biochemical methods. The results imply, that (+)-cyanidanol-3 is probably protecting the plasma membrane of hepatocytes and therefore reduces the entrance of phalloidin into the cytoplasm. In addition an increased activity of the reticuloendothelial system was observed, perhaps resulting from the flavonoid administration. Both effects could be discussed as mechanisms of flavonoid action in the liver.

[On the molecular mechanism of action of (+)-cyanidanol-3 (author's transl)]. / Zum molekularen Wirkungsmechanismus von (+)-Cyanidanol-3.

The pharmacological actions of (+)-cyanidanol-3 [(+)-flavan-3,3',4',5,7-pentol, (+)-catechin, Catergen) in various experimental liver diseases are described according to the recent available literature. On this basis the hepatoprotective activity of the drug is due to its stabilizing effect on biological membranes and its specific inhibitory activity on the Fe-ADP-, CCl4- and CBrCl3- stimulated lipoperoxidation in vitro and in vivo.

Ornithine decarboxylase activity in rat liver during phalloidin poisoning.

Rat liver ornithine decarboxylase activity was measured during phalloidin poisoning of 17 day-old rats, adult rats, and adult rats protected against phalloidin toxicity by pretreatment with somatotropin. The results show: 1. In adult rats, after a short period of activation, ornithine decarboxylase activity decreases to basal levels after phalloidin administration and remains so until death. 2. In somatotropin-pretreated rats, the enzyme activity increases with a peak 4 hours after injection of somatotropin but then drops rapidly to control levels. All rats survive poisoning. In somatotropin-pretreated rats that has also been given the ornithine decarboxylase inhibitor, 1,3-diaminopropane, phalloidin poisoning causes a decrease in the enzyme activity to basal levels and death of all animals. 3. In 17 day-old rats, ornithine decarboxylase activity decreased initially after phalloidin poisoning but then returned to unpoisoned levels. All rats survived poisoning, even those pretreated with 1,3-diaminopropane.

[Glucose tolerance and insulin sensitivity of rats poisoned with amanita-phalloidines]. / Tolerantnost' k gliukoze i chuvstvitel'nost' k insulinu u krys, otravlennykh amanita-falloidinami.

The effect Amanita phalloides on glucose tolerance and insuline sensitivity was studied. Amanita phalloides toxins were injected to albino male rats intraperitoneally in a dose of DL50. Amanita phalloides proved to cause disturbance of glucose tolerance, increased tissue glucose utilization, and enhanced the organism insuline sensitivity. The mentioned effects result from a decrease of insuline-inactivating capacity of the liver and from enhanced function of the insuline apparatus of the pancreas.

The influence of ethanol pretreatment on the effects of nine hepatotoxic agents.

The hepatotoxic effects of carbon tetrachloride (0.01 ml/kg i.p.), thioacetamide (50 mg/kg intraperitoneally), paracetamol (0.5 g/kg intraperitoneally), and allyl alcohol (0.05 ml/kg intraperitoneally) as estimated by determination of serum enzyme activities (GOT, GPT, SDH) were enhanced in mice treated with one oral dose of 4.8 g/kg ethanol 16 hrs. previously. Pretreatment of mice with ethanol did not increase the hepatotoxic actions of bromobenzene (0.25 ml/kg intraperitoneally), phalloidin (1.5 mg/kg intraperitoneally), alpha-amanitin (0.75 mg/kg intraperitoneally), and praseodymium (12 mg/kg intravenously) though there was a trend to higher enzyme activities in the case of bromobenzene. In guinea-pigs ethanol also aggravated CCl4-induced liver damage, but only strengthened the hepatotoxic activity of D-galactosamine (150 mg/kg intraperitoneally). Treatment with 4.8 g/kg ethanol did not influence liver glutathione levels in mice but increased aniline hydroxylation in the 9000 x g liver homogenate supernatant of mice and guinea-pigs. A dose of 2.4 g/kg ethanol, on the other hand, neither increased aniline hydroxylase activity nor enhanced carbon tetrachloride-induced hepatotoxicity in mice. It is assumed that the enhanced sensitivity to hepatotoxic agents after treatment with ethanol may be due to an enhanced microsomal activation of these substances.

[Somatotropin, antidot against phalloidin (author's transl)]. / Somatotropin, Antidot gegen Phalloidin.

1. Somatotropin protects rats against a lethal dose of phalloidin (1.3 mg/kg). 2. Scanning electron microscopy has shown that 2 hours after phalloidin injection the liver from a somatotropin-pretreated rat is not significantly different to that from an untreated rat. Phalloidin alone caused complete destruction of the structure of the liver lobules. 3. Somatotropin does not prevent phalloidin uptake by the liver but slows down elimination. 4. The findings are discussed with respect to their therapeutic possibilities as somatropin protects rats against death also after phalloidin poisoning.