Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Dynamic changes of acoustic load and complex impedance as reporters for the cytotoxicity of small molecule inhibitors.

Cellular motility is the major driving force of numerous biological phenomena including wound healing, immune response, embryogenesis, cancer formation, and metastasis. We studied the response of epithelial FaDu monolayers cultured on gold electrodes of an acoustic resonator (quartz crystal microbalance, QCM) and impedance sensor (electric cell-substrate impedance sensing, ECIS) to externally applied chemical stimuli interfering with cytoskeleton organization. Epithelial cell motility of confluent monolayers is characterized by subtle cell shape changes and variations in the cell-substrate as well as cell-cell distance without net directionality of individual cells. The impact of small molecules such as cytochalasin D, phalloidin, and blebbistatin as well as paclitaxel, nocodazol, and colchicin on actin and microtubules organization was quantified by conventional sensors' readouts and by comparing the noise pattern of the signals which is attributed to cellular dynamics. The responsiveness of noninvasive and label-free techniques relying on cellular dynamics is compared to classical viability assays and changes of the overall impedance of ultrasmall electrodes or acoustic loads of a thickness shear mode resonator. Depending on the agent used, a distinct sensor response was found, which can be used as a fingerprint of the cellular response. Cytoskeletal rearrangements and nuclear integrity were corroborated by fluorescence microscopy and correlated to the readouts of QCM and ECIS.

The effect of toxins on inorganic phosphate release during actin polymerization.

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity.

Phalloidin causes severe liver damage characterized by marked cholestasis, which is due in part to irreversible polymerization of actin filaments. Liver uptake of this toxin through the transporter OATP1B1 is inhibited by the bile acid derivative BALU-1, which does not inhibit the sodium-dependent bile acid transporter NTCP. The aim of the present study was to investigate whether BALU-1 prevents liver uptake of phalloidin without impairing endogenous bile acid handling and hence may have protective effects against the hepatotoxicity induced by this toxin. In anaesthetized rats, i.v. administration of BALU-1 increased bile flow more than taurocholic acid (TCA). Phalloidin administration decreased basal (-60%) and TCA-stimulated bile flow (-55%) without impairing bile acid output. Phalloidin-induced cholestasis was accompanied by liver necrosis, nephrotoxicity and haematuria. In BALU-1-treated animals, phalloidin-induced cholestasis was partially prevented. Moreover haematuria was not observed, which was consistent with histological evidences of BALU-1-prevented injury of liver and kidney tissue. HPLC-MS/MS analysis revealed that BALU-1 was secreted in bile mainly in non-conjugated form, although a small proportion (<5%) of tauro-BALU-1 was detected. BALU-1 did not inhibit the biliary secretion of endogenous bile acids. When highly choleretic bile acids, - ursodeoxycholic (UDCA) and dehydrocholic acid (DHCA) - were administered, they were found less efficient than BALU-1 in preventing phalloidin-induced cholestasis. Biliary phalloidin elimination was low but it was increased by BALU-1>TCA>DHCA>UDCA. In conclusion, BALU-1 is able to protect against phalloidin-induced hepatotoxicity, probably due to an inhibition of the liver uptake and an enhanced biliary secretion of this toxin.

Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells.

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover, depolymerizing actin reduced FAK, p38 and JNK activation during OB differentiation of hMSCs, while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation.

Temperature dependence of phalloidin response in isolated rat hepatocytes.

Below 21 degrees C isolated hepatocytes are insensitive to phalloidin. Between 21 and 30 degrees C the amount of affected cells increases to nearly maximal values, whereas a further rise of the temperature has little additional effect. These findings suggest that phase transition of membranal lipids might be responsible for the unusual temperature characteristics. The fluidity of lipids seems to control an early step of the phalloidin response.

Is ligandin relevant for the uptake and storage of phallotoxins in liver cells?

To exclude an involvement of ligandin in the uptake and storage of phalloidin in hepatocytes equilibrium-dialysis studies were made with phalloidin, cholic acid and bromosulfophthalein (BSP). Binding studies with isolated ligandin indicated that the affinity of ligandin for phalloidin is low (KD = 0.8 X 10-3 M). Phalloidin neither displaced BSP (KD = 1.3 X 10-7 M) or cholic acid (KD = 7.6 X 10-5 M) from ligandin, when preloaded with these substrates. Hepatocytes prepared from rats after daily treatment with phenobarbital during 5 days contained 3-4-fold concentrations of ligandin and bound greater amounts of BSP than controls, Nevertheless the velocity of the uptake both of [3H]-demethylphalloin ([3H]-DMP) and of [35S]-BSP was not augmented. Also the sensitivity of liver cells to phalloidin was not drastically modified after induction with phenobarbital and agrees with earlier findings in vivo. We conclude that ligandin plays a negligible role in the uptake and a minor role in a storage of phallotoxins in liver cells.

Effect of androgens on phalloidin-induced liver toxicity in mice.

A single i.p. dose of phalloidin, 0.75 or 1 mg/kg body weight, induced peliosis hepatis-like lesion (PHLL) at 3 h after injection to mice. Pretreatment with various doses of testosterone propionate for 7 days enhanced phalloidin-induced PHLL. Other anabolic steroids, methyltestosterone, methandriol. fluoxymesterone and oxymetholone had no effect.

The biochemical studies on phalloidin-induced cholestasis in rats.

We investigated sequential changes in bile flow, serum and biliary biochemical parameters in phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration with phalloidin (500 microg/kg) for 7 days, and then the animals were allowed to survive for 1, 2, 4, 7, 14 and 28 days after the last treatment. In phalloidin-treated rats, bile flow significantly decreased up to 4 days of recovery, compared with the control animals. In contrast, serum ALP activity, LAP activity, cholesterol concentration and phospholipid concentration exhibited a marked elevation throughout the recovery periods. For biliary parameters, bilirubin excretion rate was unchanged but, cholesterol excretion rate showed a marked decrease throughout the recovery periods. These results demonstrate that some parameters, particularly important indexes of cholestasis (serum ALP, cholesterol, bile flow and so on), continued significant changes at least 4 days after the last administration of phalloidin. These results demonstrate that successive treatment with phalloidin can cause damage in most of serum and biliary parameters at a chronic stage of cholestasis. Thus, our findings may provide useful information for diagnosis of drug-induced cholestasis and help to further elucidate the biochemical mechanisms of drug-induced cholestasis in humans.

Phalloidin hepatotoxicity in rats in vivo. Effect of a sympatholytic agent: propranolol.

Phalloidin, one of the main toxins of Amanita phalloides, induced hepatotoxicity in female Wistar rats at 0.9 mg/kg dose i.p. Biliary secretion was selectively inhibited after 3h, but was restored after 24 h. Phalloidin also induced a cytolytic lesion, but not a fatty liver, as in alpha-amanitin intoxication. Propranolol pretreatment (30 min prior to phalloidin injection) did not afford protection against hepatotoxicity, but increased alkaline phosphatase, 5'-nucleotidase and aminotransferase activities.

Protection by indomethacin against the lethality and hepatotoxicity of phalloidin in mice.

The present study examined the possible involvement of endogenous cyclooxygenase-derived factors in the lethality and hepatic hemorrhagic necrosis induced by phalloidin. Mice were pretreated with indomethacin, aspirin or ibuprofen (all inhibitors of cyclooxygenase) and injected with phalloidin (2 mg/kg). The toxin induced 75% lethality and caused severe hemorrhagic necrosis of the liver associated with increased serum levels of AST and ALT. Indomethacin completely prevented the mortality and hepatic damage elicited by phalloidin as judged by morphologic analysis and serum AST and ALT release. The in vitro addition of indomethacin to suspensions of freshly-isolated hepatocytes decreased plasma membrane bleb formation induced by phalloidin. In contrast to indomethacin, aspirin and ibuprofen did not influence phalloidin toxicity in vivo. These results suggest that inhibition of prostanoids per se may not be the sole mechanism of protection by indomethacin.

[A review of mushrooms containing amanitins and phalloidines]. / Prehled hub obsahujících amanitiny a faloidiny.

The toxic peptides amanitins and phalloidins were found in 22 species of gilled fungi in Europe: 3 species of the Amanita genus, 8 species of the Galerina genus and 11 of the Lepiota genus. Galerina sulcipes in the most toxic one, followed by Amanita phalloides, A. virosa, Galerina marginata and Lepiota brunneoincarnata.

[Antimitotic compounds as modifiers of the radiation lesion of cultured cells]. / Antimitoticheskie soedineniia kak modifikatory luchevogo porazheniia kul'tiviruemykh kletok.

The features of antimitotic substances as radioprotectors were studied. In vitro experiments have demonstrated that taxol revealed radioprotective features concerning the process of polymerization of irradiated microtubules. These results were the basis for the use of taxol and some other substances with high affinity for cytoskeleton proteins as potential radiomodificators in vivo. Experiments with cultivated fibroblasts revealed that colchicine significantly enhances radioactive injuries of cells while taxol and phalloidin manifest their radioprotective features.

Stabilization of actin filaments prevents germinal vesicle breakdown and affects microtubule organization in Xenopus oocytes.

In Xenopus oocytes, extremely giant nuclei, termed germinal vesicles, contain a large amount of actin filaments most likely for mechanical integrity. Here, we show that microinjection of phalloidin, an F-actin-stabilizing drug, prevents the germinal vesicle breakdown (GVBD) in oocytes treated with progesterone. These nuclei remained for more 12 h after control oocytes underwent GVBD. Immunostaining showed significant elevation of actin in the remaining nuclei and many actin filament bundles in the cytoplasm. Furthermore, microtubules formed unusual structures in both nuclei and cytoplasm of phalloidin-injected oocytes stimulated by progesterone. Cytoplasmic microtubule arrays and intranuclear microtubules initially formed in phalloidin-injected oocytes as control oocytes exhibited white maturation spots; these structures gradually disappeared and finally converged upon intranuclear short bundles when control oocytes completed maturation. In contrast, treatment of oocytes with jasplakinolide, a cell membrane-permeable actin filament-stabilizing drug, did not affect GVBD. This drug preferentially induced accumulation of actin filaments at the cortex without any increase in cytoplasmic actin staining. Based on these results, intranuclear and cytoplasmic actin filament dynamics appear to be required for the completion of GVBD and critically involved in the regulation of microtubule assembly during oocyte maturation in Xenopus laevis.

Characterization of organic anion transporting polypeptide 1b2-null mice: essential role in hepatic uptake/toxicity of phalloidin and microcystin-LR.

The liver-specific importer organic anion transporting polypeptide 1b2 (Oatp1b2, Slco1b2, also known as Oatp4 and Lst-1) and its human orthologs OATP1B1/1B3 transport a large variety of chemicals. Oatp1b2-null mice were engineered by homologous recombination and their phenotype was characterized. Oatp1b2 protein was absent in livers of Oatp1b2-null mice. Oatp1b2-null mice develop normally and breed well. However, adult Oatp1b2-null mice had moderate conjugated hyperbilirubinemia. Compared with wild-types, Oatp1b2-null mice had similar hepatic messenger RNA expression of most transporters examined except a higher Oatp1a4 but lower organic anion transporter 2. Intra-arterial injection of the mushroom toxin phalloidin (an Oatp1b2-specific substrate identified in vitro) caused cholestasis in wild-type mice but not in Oatp1b2-null mice. Hepatic uptake of fluorescence-labeled phalloidin was absent in Oatp1b2-null mice. Three hours after administration of microcystin-LR (a blue-green algae toxin), the binding of microcystin-LR to hepatic protein phosphatase 1/2a was much lower in Oatp1b2-null mice compared with wild-type mice. In contrast, Oatp1b2-null mice were transiently protected from decrease in bile flow induced by estradiol-17beta-D-glucuronide, a common substrate for Oatps. Oatp1b2-null mice were completely resistant to the hepatotoxicity induced by phalloidin and microcystin-LR, but were similarly sensitive to alpha-amanitin-induced hepatotoxicity compared with wild-type mice. In conclusion, Oatp1b2-null mice display altered basic physiology and markedly decreased hepatic uptake/toxicity of phalloidin and microcystin-LR. Oatp1b2-null mice are useful in elucidating the role of Oatp1b2 and its human orthologs OATP1B1/1B3 in hepatic uptake and systemic disposition of toxic chemicals and therapeutic drugs.

Effects of phalloidin on hepatic gene expression in mice.

An attempt has been made to identify molecular markers of intrahepatic cholestasis in mice employing phalloidin as a cholestatic agent. Phalloidin was administered to BALB/c mice at three predetermined dose: 250 microg/kg, 500 microg/kg, and 1 mg/kg for 1, 3, and 7 days. Liver function was estimated to confirm cholestasis. Histopathological observations on liver were also made to confirm liver injury. Phalloidin at 1 mg/kg for 7 days was found to induce cholestasis. Therefore gene expression studies were confined to this group only. A total of 88 genes were found to be affected by phalloidin. These were the genes associated with cytoskeleton regulation as well as tight junction, focal adhesion, and ATP-binding cassette transporters. Such proteins obstruct the removal of bile components from hepatocytes to the bile canaliculus or blood. Phalloidin treatment did not affect the proteins responsible for cell maintenance or death. The authors show that phalloidin-induced intrahepatic cholestasis is manifested by disturbing the cytoskeleton. The set of genes up-regulated by phalloidin can be considered as molecular markers of intrahepatic cholestasis. The observations are further expected to be helpful in the management of cholestatic pharmaceuticals and associated problems of liver diseases in humans.