W. Thomas Shier

THE FUMONISIN PARADOX: A REVIEW OF RESEARCH ON ORAL BIOAVAILABILITY OF FUMONISIN B1, A MYCOTOXIN PRODUCED BY FUSARIUM MONILIFORME

The fumonisins are a series of mycotoxins produced by Fusarium moniliforme, a ubiquitous contaminant of stored corn (maize) worldwide. Consumption of food products contaminated with F. moniliforme hasbeen correlated with increased risk of human esophageal cancer in epidemiological studies in southern Africa and China. The most abundant component, fumonisin B1 (FB1), was isolated from F. moniliforme culture extracts using a short-term tumor promoter bioassay to guide the fractionation. Purified FB1 has been confirmed to act as a tumor promoter in animal model systems; to cause hepatocellular carcinoma, cirrhosis and proximal tubule nephrosis in rats; and to mediate agriculturally significant diseases associated withconsumption of F. moniliforme-contaminated feeds, including equine leukoencephalomalacia and porcine pulmonary edema. However, studies on the toxicokinetics of radiolabeled and unlabeled FB1 carried out by three research groups in five animal species all indicate that it is absorbed very poorly if at all when administered orally. There is no evidence for functionally significant metabolism of FB1 in vivo. These observations result in what might be called the “fumonisin paradox”—how can the toxin cause agriculturally significant diseases and possibly human cancer if it is not effectively adsorbed after oral administration? There are several plausible explanations including (i) an unknown, readily bioavailable contaminating toxin is responsible; (ii) higher FB1 bioavailability at lower dose; (iii) greater conversion to active metabolites at lower dose; (iv) bioaccumulation and (v) effective uptake of FB1 derivatives that are readily converted back to FB1 or active metabolites in the body. The full extent of the threat to food safety posed by the fumonisins will not be known until the factors affecting oral bioavailability are understood.

Selection of Aspergillus flavus isolates for biological control of aflatoxins in corn

The fungus Aspergillus flavus is responsible for producing carcinogenic mycotoxins, the aflatoxins, on corn (maize) and other crops. An additional harmful toxin, cyclopiazonic acid, is produced by some isolates of A. flavus. Several A. flavus strains that do not produce one or both of these mycotoxins are being used in biological control to competitively exclude the toxin-producing strains from the agroecosystem, particularly from seeds, grain and other marketable commodities. Three well-studied non-aflatoxigenic strains, including two that are commercially available, have been compared in side-by-side field trials. The results of that study, together with a growing understanding of A. flavus ecology and new genetic insights, are guiding the selection of biocontrol strains and influencing crop management decisions for safe and sustainable production.

Dynamics of Mycotoxin and Aspergillus flavus Levels in Aging Bt and Non-Bt Corn Residues under Mississippi No-Till Conditions

Mycotoxin and Aspergillus flavus levels in soil-surface corn debris left by no-till agriculture methods (stover, cobs, and cobs with grain) were determined during the December-March fallow period for near-isogenic Bt and non-Bt hybrid corn. By December, average mycotoxin levels in non-Bt corn were many times higher in cobs with grain than in grain harvested in September (total aflatoxins, 774 vs 211 ng/g; total fumonisins, 216 vs 3.5 microg/g; cyclopiazonic acid, 4102 vs 72.2 microg/g; zearalenone, 0.2 vs < 0.1 microg/g). No trichothecenes were detected. Levels of mycotoxins and A. flavus propagules were approximately 10- to 50-fold lower in cobs without grain and stover, respectively, for all mycotoxins except zearalenone. Mycotoxin levels in corn debris fractions decreased during winter but began to rise in March. Levels of all mycotoxins and A. flavus propagules were lower in harvested grain and debris from Bt than non-Bt corn, but differences were significant (p < 0.05) only for aflatoxins.

Complete structures of the sphingosine analog mycotoxins fumonisin B1 and AAL toxin TA: Absolute configuration of the side chains

Abstract Fumonisin B 1 and AAL toxin T A are sphingosine-analog mycotoxins characterized by propane-1,2,3-tricarboxylic acid side chains esterified to alkylamine backbones. The absolute configuration of all stereogenic centers in the backbones is known. Using chiral gas chromatography methodology we have determined the absolute configuration at C-3′ in the side chains to be S, thereby completing structure determination of both toxins.

Polycations as prostaglandin synthesis inducers: Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(l-lysine) and other synthetic polycations

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.

Antimalarial, antiviral, and antitoxoplasmosis norsesterterpene peroxide acids from the Red Sea sponge Diacarnus erythraeanus.

A new norsesterterpene acid, named muqubilone (1), along with the known sigmosceptrellin-B and muqubilin were isolated from the Red Sea sponge Diacarnus erythraeanus. The structure determination of 1 was based primarily on 1D and 2D NMR analyses. Sigmosceptrellin-B exhibits significant in vitro antimalarial activity against Plasmodium falciparum (D6 and W2 clones) with IC(50) values of 1200 and 3400 ng/mL, respectively. Muqubilin and 1 show in vitro antiviral activity against herpes simplex type 1 (HSV-1) with ED(50) values of 7.5 and 30 microg/mL, respectively. Muqubilin and sigmosceptrellin-B display potent in vitro activity against Toxoplasma gondii at a concentration of 0.1 microM without significant toxicity.

Sodium- and calcium-de-endent steps in the mechanism of neonatal rat cardiac myocyte killing by lonophores. I. The sodium-carrying lonophore, monensin

Toxicosis by monensin, a Na(+)-selective ionophore, induces skeletal and cardiac muscle necrosis. Cultured neonatal rat cardiac myocytes were killed by monensin (greater than 0.2 micrograms/ml) beginning at 30 min and completing by 60-90 min. Other cultured cell types presumably lacking excitable membranes were not killed by monensin under these conditions. Cardiac myocytes were also killed by nigericin and nonactin (monovalent cation-carrying ionophores with low ion selectivity), but not by valinomycin, which has a high selectivity for K+. Monensin-induced killing was associated with formation of blebs in cell membranes and subsequent swelling of the cells during the early phases of killing, whereas surface membranes of cells permeabilized to trypan blue dye contained discrete small holes visible by scanning electron microscopy. Monensin-induced killing occurred at extracellular Na+ concentrations greater than or equal to 10 mM, but not when Li+, K+, Cs+, Rb+, or choline ions replaced Na+ at concentrations up to 0.15 M. Killing was prevented at extracellular pH values less than or equal to 6.4 and was enhanced by ouabain, an inhibitor of Na+/K(+)-ATPase-mediated Na+ transport. Several characteristics of monensin-induced cardiac myocyte killing were similar to those observed during killing induced by the Ca(2+)-carrying ionophore, A23187 plus Ca2+, including a requirement for extracellular Ca2+ concentrations greater than 0.5 mM, inhibition by Mn2+ and Ni2+, and an associated stimulation of arachidonic acid release. The cell killing characteristics are consistent with a monensin-induced Na+ influx which admits toxic levels of extracellular Ca2+ to the cytoplasm of cells with excitable membranes, possibly via Na+/Ca2+ antiporter protein(s).

Sphingosine Analogs: an Emerging New Class of Toxins that Includes the Fumonisins

AbstractThere is an emerging new class of toxins - the sphingosine analog toxins. First to be identified were the AAL toxins, host-specific toxins produced by the fungus Alternaria alternata f. sp. lycopersici to facilitate invasion of tissues in susceptible plants. The best studied members of the class are the fumonisins, which were initially isolated as tumor promoters from corn infested with Fusarium moniliforme, the major ear rot fungus of corn and an important pathogen of stored grains worldwide. Recently, the sphingosine analog toxins penaresidins A and B and penazetidine A have been isolated from marine sponges of the Penares genus. All these toxins are long-chain fatty amines with free amino groups. The mycotoxins are characterized by the absence of a C-1 hydroxyl group and the presence of one or more propanetricarboxylic acid esters, whereas the sponge toxins are characterized by a four-membered azetidine ring. The observation that removing propanetricarboxylic acid moieties from fumonisins by al...

Studies on the mechanisms of mammalian cell killing by a freeze-thaw cycle: Conditions that prevent cell killing using nucleated freezing

Normally a freeze-thaw cycle is a very efficient method of killing mammalian cells. However, this report describes conditions that prevent killing of cultured mammalian cells by nucleated freezing at -24 degrees C. Optimal protection from cell killing at -24 degrees C was obtained in isotonic solutions containing an organic cryoprotectant such as dimethyl sulfoxide (DMSO; 10%, v/v), a saccharide such as sucrose over a broad concentration range from 50 to 150 mM, and glucose. Glycerol was also an effective cryoprotectant but other organic solvents were ineffective, although in some cases they appeared to protect cell membranes, while not protecting other vital components. A wide variety of saccharide structures were effective at protecting cells from freeze-thaw killing, with trehalose being particularly effective. The degree of resistance to killing by a freeze-thaw cycle under these conditions varied widely among different cell lines. If toxicity of DMSO was responsible for this variability of cryoprotection, it must have been due to short-term, not longer term, toxicity of DMSO. Studies on the mechanism by which cells are protected from killing under these conditions indicated that neither vitrification of the medium nor the concentrating of components during freezing were involved. One model not eliminated by the mechanistic studies proposes that the organic solvent cryoprotectant component acts by fluidizing membranes under the thawing conditions, so that any holes produced by ice crystals propagating through membranes can reseal during the thawing process. In this model one of the mechanisms by which the saccharide component could act is by entering the cells and stabilizing vital intracellular components. Consistent with this, a freeze-thaw cycle promoted the uptake of labeled sucrose into cultured cells.

Phospholipase activation in the cytotoxic mechanism of thionin purified from nuts of Pyrularia pubera

Treating NIH3T3 fibroblast cells with Pyrularia pubera thionin (100 micrograms/ml) stimulated release of labelled free fatty acids from phospholipids biosynthetically labelled by incorporation of [3H]arachidonic acid. Since Pyrularia thionin exhibited no detectable phospholipase activity, it was concluded that the release response represented activation of endogenous phospholipases in the cells. The phospholipase activated by Pyrularia thionin (100 micrograms/ml) stimulated the release of 61% of the incorporated [3H]arachidonate in the presence of 1.8 mM extracellular calcium with maximum activation at 90 min following an initial lag period of about 20 min. The release response exhibited little dependence on extracellular calcium at this thionin concentration, but at concentrations 20 micrograms/ml, extracellular calcium appeared to be inhibitory to phospholipase activation. Some characteristics of the fatty acid release response are consistent with activation of a lysosomal phospholipase being part of the cellular response to Pyrularia thionin. Activation of a lysosomal enzyme can occur independently or as a result of coordinate activation of the whole lysosome, which would expose other cellular components of degradative lysosomal enzymes. Consistent with coordinate activation of lysosomal enzymes, Pyrularia thionin also stimulates the production of small, trichloroacetic acid-soluble peptides and nucleic acid fragments from biosynthetically-labelled RNA and proteins in treated cells. It is not clear from the results obtained what role, if any, activation of lysosomal enzymes plays in the overall toxic response to Pyrularia thionin in NIH3T3 cells. However, Pyrularia pubera thionin may represent a useful tool for studying the regulation of lysosomal enzymes and their roles in cells.