Induction of apoptosis by fumonisin B1 in HT29 cells is mediated by the accumulation of endogenous free sphingoid bases.

Fumonisin B1 (FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains of Fusarium moniliforme. Incubation of HT29 cells (a human colonic cell line) with FB1 or AP1 caused a significant reduction in cell number; AP1 was less potent, with 50 microM AP1 causing the same reduction (ca. 30% after 24 h) as 10 microM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1 and AP1 caused the accumulation of sphinganine (25- and 35-fold by 10 microM FB1 and 50 microM AP1, respectively); thus, concentrations of FB1 and AP1 that caused comparable reductions in cell number were also similar with respect to elevation of sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1 on HT29 cells can be attributed to the accumulation of sphinganine. Since consumption of food contaminated with Fusarium moniliforme (Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1 and AP1 are toxic for intestinal cells in vivo should be evaluated, especially in the light of the recent report (Bhat et al., Clin. Toxicol. 35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins.

Sphingosine and sphinganine levels in human mesothelial cells in vitro as a potential index of signal transduction pathways impacted by microbes and osmolality.

Sphingolipids are emerging as important regulators of mammalian cell biology. In this study, the contents of six separate preparations of human omental mesothelial cells in vitro were examined for free sphingosine and sphinganine, and for the total levels of these sphingoid bases in ceramide-containing sphingolipids. Two high-performance liquid chromatography (HPLC) methods for determination of sphingoid base levels in cultured cells were compared. The rapid-HPLC method was found to yield the highest recovery of internal standard. Mesothelial cells initially isolated by collagenase digestion of the omentum were found to have higher free- and total-sphingoid base levels than cells isolated by trypsin-EDTA digestion. Use of sphingoid base levels to gain insights into the status of cellular nutrition, inflammation, programmed cell death, exposure to microbial toxins, cytokines, and growth factors within the peritoneum will require a systematic description of sphingolipids in normal, diseased, and dialyzed mesothelium.

Mycotoxin-induced elevation of free sphingoid bases in precision-cut rat liver slices: specificity of the response and structure-activity relationships.

Fumonisin B1 (FB1) is the predominant member of a family of toxic metabolites produced by several species of Fusarium and is commonly found on corn. FB1 is a potent competitive inhibitor of ceramide synthase, which catalyzes the conversion of sphinganine and sphingosine to ceramide. The resultant accumulation of free sphingoid bases and the disruption of sphingolipid metabolism is believed to be the mechanism of toxicity of the fumonisins. The objectives of this study were to determine the relative potency of analogs of FB1 to inhibit ceramide synthase and to determine whether the inhibition is specific to mycotoxins with fumonisin-like structures. Fumonisins B1, B2, B3, B4, C4, and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f. sp. lycopersici) were approximately equipotent inhibitors. Hydrolyzed fumonisins B1, B2, and B3, which lack the tricarballylic side chains, were only 30-40% as potent as the parent toxins. N-acetylated FB1 (FA1) did not block ceramide synthase, suggesting that FA1 is nontoxic. Inhibition of ceramide synthase by fumonisin analogs did not appear to be related to the lipophilicity of the compounds, as determined by computer estimation of log P values. The ability of relatively high (10 and 100 microm) doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B1, cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and zearalenone, to block ceramide synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response.

Polycystic kidney disease: an unrecognized emerging infectious disease?

Polycystic kidney disease (PKD) is one of the most common genetic diseases in humans. We contend that it may be an emerging infectious disease and/or microbial toxicosis in a vulnerable human subpopulation. Use of a differential activation protocol for the Limulus amebocyte lysate (LAL) assay showed bacterial endotoxin and fungal (1-->3)-beta-D-glucans in cyst fluids from human kidneys with PKD. Fatty acid analysis of cyst fluid confirmed the presence of 3-hydroxy fatty acids characteristic of endotoxin. Tissue and cyst fluid from three PKD patients were examined for fungal components. Serologic tests showed Fusarium, Aspergillus, and Candida antigens. IgE, but not IgG, reactive with Fusarium and Candida were also detected in cyst fluid. Fungal DNA was detected in kidney tissue and cyst fluid from these three PKD patients, but not in healthy human kidney tissue. We examine the intertwined nature of the actions of endotoxin and fungal components, sphingolipid biology in PKD, the structure of PKD gene products, infections, and integrity of gut function to establish a mechanistic hypothesis for microbial provocation of human cystic disease. Proof of this hypothesis will require identification of the microbes and microbial components involved and multifaceted studies of PKD cell biology.

An optimized MTT bioassay for determination of cytotoxicity of fumonisins in turkey lymphocytes.

In vitro cytotoxicity assays have been performed for detection and quantitation of fumonisins, as possible alternatives for whole animal testing. This study was undertaken to establish optimal in vitro conditions using turkey lymphocytes. Turkey lymphocytes were isolated from peripheral blood by Percoll gradient centrifugation. Cytotoxicity of fumonisin B1 (FB1) and B2 (FB2) was determined by exposing lymphocytes to FB1 or FB2 at concentrations of 0.01-25 micrograms/mL for 24, 48, or 72 h at 39 degrees C. The MTT bioassay was used to measure cell viability and proliferation. In metabolically active cells, the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], was reduced to MTT formazan. Turkey lymphocytes that had been exposed in vitro to FB1 and FB2 for 48 and 72 h showed inhibition of cell proliferation that was dose-dependent. The 50% inhibitory dose for FB1 and FB2 was 0.4-5 micrograms/mL. Cells exposed to FB1 or FB2 exhibited high levels of cytoplasmic vacuolization and were unable to proliferate, whereas proliferation of control lymphocytes was observed at 48 and 72 h. FB2 was 3- to 4-fold more cytotoxic than FB1.

Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin from Fusarium proliferatum.

Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclaved Fusarium proliferatum culture material containing fumonisin B1 (FB1), fumonisin B2 (FB2) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61-546 ppm FB1, 14-94 ppm FB2 and 66-367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended with F. proliferatum culture material containing FB1, FB2 and moniliformin.