antamanide and phallacidin

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 β-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.

Topics: Amanita; Amanitins; Chromatography, Liquid; Humans; Mass Spectrometry; Mushroom Poisoning; Peptides, Cyclic; Phalloidine; Poisons

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

Topics: Amanitins; Animals; Binding, Competitive; Biological Transport; Cells, Cultured; Cholic Acids; Epoprostenol; Estradiol; Kinetics; Liver; Monensin; Oligopeptides; Peptides, Cyclic; Phalloidine; Prostaglandins F, Synthetic; Rats; Silybin; Silymarin; Somatostatin; Taurocholic Acid; Verapamil