phalloidine and antamanide

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

In order to investigate the antiproliferative properties of antamanide, we have synthesized and studied two antamanide analogs where the phenylalanine residue in positions 6 or 9 is substituted by tyrosine, their corresponding linear forms and the cyclic and linear des Phe5,Phe6-Tyr9-analogs. Antamanide and its biologically active synthetic analogs are able to form highly stable complexes with metal ions, particularly Na+, K+ and Ca2+. We studied the ion-binding properties of the Tyr-antamanide analogs by CD and Tb3+ -mediated fluorescence in acetonitrile. In this medium the far-and near-UV CD spectra of the neat Tyr6-antamanide analog are very similar to that of the parent cyclic decapeptide. Substantial differences occur on the contrary in the CD spectra of the neat Tyr9-antamanide, particularly in the regions at 220 nm and 270-290 nm. In acetonitrile, as already found for antamanide, the interaction with the above-mentioned metal ions always produces evident changes in the far- and near-UV CD spectra of both analogs. On the contrary, the CD spectra of the linear deca- and octa- and of the cyclic octa-analogs are affected by the presence of metal ions only in the near-UV region. In the same solvent the Tb3+ -mediated fluorescence spectra of all the synthetic peptides are remarkably affected by the addition of ions. On the basis of the spectral total changes, by using either or both the spectroscopic techniques, it has been possible to determine the ion binding constants for all the linear and cyclic Tyr-antamanide analogs and to compare them with that of the parent peptide. The antitoxic and antiproliferative activities of these antamanide analogs have been tentatively correlated to their ion-binding properties. A preliminary account of this work was given in (1).

Topics: Acetonitriles; Amino Acid Sequence; Amino Acid Substitution; Animals; Antidotes; Calcium; Cations; Cell Division; Circular Dichroism; Male; Melanoma; Metals; Metals, Rare Earth; Mice; Peptide Fragments; Peptides, Cyclic; Phalloidine; Potassium; Sodium; Spectrometry, Fluorescence; Terbium; Tumor Cells, Cultured

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Animals; Blood Proteins; Capillary Permeability; Cytochalasin D; Dogs; Endothelium, Vascular; Female; Ischemia; Leukocyte Adherence Inhibition Test; Male; Muscles; Neutrophils; Peptides, Cyclic; Peroxidase; Phalloidine; Reperfusion; Superoxides

Interleukin 2 (IL-2) is a potent cytokine with diverse effects, including the ability to stimulate lymphocyte differentiation into cells capable of lysing tumor. Its therapeutic efficacy is limited because of side effects such as breakdown of the microvascular barrier and edema. Control of the microvascular barrier is in part regulated by endothelial cell cytoskeletal contractile proteins. This study tests whether the cyclopeptides that maintain actin filament organization and distribution and reduce macromolecular flux across the endothelial cell junction in vitro would similarly maintain barrier tightness and prevent early edema produced by IL-2 in vivo. Anesthetized rats were treated at 30-min periods with intravenous saline (0.5 ml, n = 41), phalloidin (20 micrograms in 0.5 ml, n = 21), or antamanide, (20 micrograms in 0.5 ml, n = 21), starting 30 min before the 1-h infusion of 10(6) U of recombinant human IL-2 or saline. Six hours after the start of IL-2, there was edema in the saline/IL-2 group, as measured by increased wet-to-dry ratios (W/D) in the lungs, heart, and kidney. With saline/IL-2, bronchoalveolar lavage (BAL) fluid contained an elevated protein concentration and higher plasma thromboxane levels compared with controls. The number of neutrophils sequestered in the lungs was more than twice that of saline controls. Phalloidin significantly attenuated edema in lung and reduced BAL protein leak. Antamanide treatment was as effective in limiting lung and heart edema, but, in contrast to phalloidin, antamanide prevented kidney edema and did not lead to an alteration in the liver W/D. Antamanide also prevented BAL fluid protein leak.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Capillary Permeability; Chemical and Drug Induced Liver Injury; Edema; Edema, Cardiac; Interleukin-2; Kidney Diseases; Liver Diseases; Male; Peptides, Cyclic; Phalloidine; Pulmonary Edema; Rats; Rats, Inbred Strains; Thromboxane B2

The effects of antamanide (10(-14)-10(-5) M) and N-acetyl-secophalloidin (10(-7)-5 X 10(-3) M) a neutral non-toxic derivative of phalloidin, were tested on voltage-clamped single frog muscle fibres. Antamanide protected muscle fibres against the negative inotropic effect of phalloidin but blocked the fast potassium permeability in the same concentration range and the same voltage-dependent manner as did phalloidin. N-Acetyl-secophalloidin exhibited a strongly attenuated blocking effect on K+ permeability in a 1,000-fold higher concentration range than phalloidin. Neither antamanide nor N-acetyl-secophalloidin affected the contractile properties. These results suggest the existence in the frog muscle membrane of a receptor with two sites for phalloidin and antamanide which acts on potassium conductance.

Topics: Animals; Depression, Chemical; In Vitro Techniques; Ion Channels; Membrane Potentials; Muscles; Myocardial Contraction; Oligopeptides; Peptides, Cyclic; Permeability; Phalloidine; Rana ridibunda

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

Topics: Amino Acid Sequence; Animals; Biological Transport; Cholic Acids; Growth Hormone; In Vitro Techniques; Liver; Molecular Conformation; Peptides, Cyclic; Phalloidine; Somatostatin; Stereoisomerism; Structure-Activity Relationship

Topics: Animals; Cell Aggregation; Female; Leukemia, Experimental; Lymphocytes; Mice; Mice, Inbred AKR; Oligopeptides; Peptides, Cyclic; Phalloidine; Pilot Projects; Prospective Studies

The synthetic analog of antamanide in which all four phenylalanyl residues are hydrogenated to cyclohexylalanyl (Cha) residues, cyclic(Val-Pro-Pro-Ala-Cha-Cha-Pro-Pro-Cha-Cha), has a complete loss of antitoxic potency despite its ability to form ion complexes in the same manner as antamanide. The conformation of Li+.perhydroantamanide has been established in the present paper by x-ray diffraction analysis of a single crystal. The backbone encapsulates a Li+ ion in an almost identical manner as in Li+ antamanide. However, in Li+ antamanide the four phenyl groups are folded against the globular backbone, thus providing a hydrophobic surface for the complex, whereas in Li+ X perhydroantamanide the four cyclohexyl moieties are extended away from the folded backbone, resulting in the exposure of large portions of the polar backbone to the environment. As a consequence, four NH groups form hydrogen bonds with Br- ions, three C--O groups form hydrogen bonds with water molecules, and one C--O group makes a ligand to an additional external Li+ ion. The internal Li+ ion is pentacoordinated, whereas the external Li+ ion is tetracoordinated. The large change of the hydrophobicity around the midsection of the perhydroantamanide complex may be related to the biological inactivity. The content per asymmetric unit of the crystal is C64H102N10O10 X 2Li+ X 4H2O X 2CH3CN.2Br- in space group P2(1)2(1)2(1) with a = 21.740(7), b = 21.566(4), and c = 17.361(4) A. The agreement factor is 8.2% for 5135 data.

Topics: Lithium; Molecular Conformation; Peptides, Cyclic; Phalloidine; Structure-Activity Relationship; X-Ray Diffraction

Phalloidin, a bicyclic heptapeptide, and antamanide, a monocyclic decapeptide from the poisonous mushroom Amanita phalloides, interact with bile-salt-binding polypeptides of the hepatocyte membrane, as demonstrated by photoaffinity labeling using the photolabile bile salt derivative 7,7,-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, either unconjugated or taurine conjugated. With the photolabile derivatives of phalloidin, N-delta-(4-[(1-azi-2,2,2-trifluoroethyl) benzoyl]-beta-alanyl)-delta-aminophalloin, (N epsilon-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]lys6)-anta manide, the same membrane polypeptides with apparent MrS of 54,000 and 48,000 were labeled as with the photolabile derivatives of unconjugated and conjugated bile salts. The presence of bile salts decreased markedly the extent of labeling of these phalloidin- and antamanide-binding polypeptides. These results indicate that hepatic uptake systems for bile salts, phallotoxins, and the cycloamanide antamanide are identical, thus explaining the organotropism of phallotoxins.

Topics: Affinity Labels; Animals; Bile Acids and Salts; Biological Transport; Carrier Proteins; Cell Membrane; Hydroxysteroid Dehydrogenases; In Vitro Techniques; Liver; Male; Membrane Glycoproteins; Oligopeptides; Peptides, Cyclic; Phalloidine; Photochemistry; Rats; Rats, Inbred Strains