Fumonisin concentration and ceramide synthase inhibitory activity of corn, masa, and tortilla chips.

Nixtamalization removes fumonisins from corn and reduces their amounts in masa and tortilla products. Fumonisin concentrations and potential toxicity could be underestimated, however, if unknown but biologically active fumonisins are present. Therefore, the relative amounts of fumonisins in extracts of fumonisin-contaminated corn and its masa and tortilla chip nixtamalization products were determined with an in vitro ceramide synthase inhibition bioassay using increased sphinganine (Sa) and sphinganine to sphingosine ratio (Sa/So) as endpoints. African green monkey kidney cells (Vero cells ATCC CCL-81) were grown in 1-ml wells and exposed to 4 microl of the concentrated extracts for 48 h. The corn extract inhibited ceramide synthase as Sa (mean = 132 pmol/well) and Sa/So (mean = 2.24) were high compared to vehicle controls (Sa = 9 pmol/well; Sa/So = 0.10). Inhibitory activity (mean Sa = 14-24 pmol/well; mean Sa/So = 0.17-0.28) of the masa and tortilla chip extracts were reduced > or = 80% compared to the corn extract. Results were corroborated in a second experiment in which Sa and Sa/So of the wells treated with masa or tortilla chip extracts were reduced > or = 89% compared to those treated with the corn extract. Masa and tortilla chip FB1 concentrations (4-7 ppm) were reduced about 80-90% compared to the corn (30 ppm) when the materials were analyzed by high-performance liquid chromatography (HPLC). Therefore, nixtamalization reduced both the measured amount of FB1 and the ceramide synthase inhibitory activity of masa and tortilla chips extracts. The results further suggest that the masa and tortilla chip extracts did not contain significant amounts of unknown fumonisins having ceramide synthase inhibitory activity.

Inhibition of sphingolipid biosynthesis decreases phosphorylated ERK2 in LLC-PK1 cells.

Fumonisin B(1) (FB(1)) is a fungal toxin produced by Fusarium verticillioides that inhibits ceramide synthase (CS), a key enzyme in the de novo sphingolipid biosynthesis pathway. In LLC-PK(1) cells, FB(1) inhibits cell proliferation and induces apoptosis, which can be prevented by inhibitors of serine palmitoyltransferase (SPT). Inhibition of SPT prevents the FB(1)-induced accumulation of free sphinganine, a precursor of ceramide biosynthesis. However, not all of the effects of FB(1) in LLC-PK(1) cells can be explained solely by the increase in free sphingoid bases. The downstream signaling pathways that are affected by FB(1)-induced disruption of sphingolipid biosynthesis are not well understood. This study determined, in LLC-PK(1) cells, changes in p42 MAP kinase (phosphorylated ERK2 [pERK2]) phosphorylation in response to various inhibitors of key enzymes of the de novo sphingolipid biosynthesis pathway (CS, SPT, and glucosylceramide synthase [GlcCer synthase]). The results show that inhibition of any of the three enzymes caused a similar decrease in the extent of phosphorylation of ERK2 with no reduction in total ERK2. The co-treatment of FB(1) (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB(1)-induced reduction in pERK2 phosphorylation, indicating that FB(1)-mediated changes in phosphorylation of pERK2 was independent of increases in free sphinganine or its metabolites or a reduction in ceramide. Nonetheless, the decrease in pERK2 phosphorylation was dependent on inhibition of de novo sphingolipid biosynthesis. Decreased pERK2 activity could contribute to the physiological effects of FB(1) in LLC-PK(1) cells that are not due to alteration in pathways modulated by free sphingoid bases and their metabolites but are sensitive to inhibition of glycosphingolipid biosynthesis.

Fumonisin-ortho-phthalaldehyde derivative is stabilized at low temperature.

Fumonisins are water soluble mycotoxins produced by the fungus Fusarium verticillioides (formerly F. moniliforme). Fumonisin B(1) (FB(1)) is a diester of propane-1,2,3-tricarboxylic acid and 2-amino-12, 16-dimethyl-3,5,10,14,15-pentahydroxyeicosane, and is the most abundant of the naturally occurring fumonisins. Upon removal of the two tricarballylic acid side chains, the structure is referred to as hydrolyzed FB(1) (HFB(1)). FB(1) and HFB(1) are structurally similar to sphinganine, a sphingoid base. The fumonisins do not absorb UV light or fluoresce; therefore, derivatizing reagents are used for detection when separation is by high performance liquid chromatography (HPLC). The standard derivatizing reagent used for HPLC is ortho-phthalaldehyde (OPA) plus 2-mercaptoethanol (ME) reaction partner, however, the OPA-FB(1) derivative is not stable at room temperature. The objectives of this study were to: (1). determine the effect of temperature on the stability of the OPA-FB(1) derivative and (2). determine which structural characteristics of FB(1) contribute to the instability of the OPA-FB(1) derivative. The results indicate that OPA-FB(1), OPA-FB(3) and OPA-HFB(1) derivatives are unstable at 24 degrees C but that their stability improves significantly at 4 degrees C. The OPA-sphinganine derivative is stable for at least 24h at 24 degrees C. Thus, the instability of the OPA-FB(1) derivative may be attributed to its lack of a hydroxyl group at the carbon 1 position.

Total fumonisins are reduced in tortillas using the traditional nixtamalization method of mayan communities.

Fumonisin B1 (FB1) is a maize mycotoxin. In tortilla preparation, maize is treated with lime (nixtamalization), producing hydrolyzed FB1 (HFB1) due to loss of the tricarballylic acid side chains. This study determined the following: 1) whether nixtamalization by Mayan communities reduces total fumonisins, and 2) the steps in the process at which reduction occurs. Tortillas prepared by the traditional process contained FB1, FB2 and FB3 and their hydrolyzed counterparts. There were equimolar amounts of FB1 and HFB1 in the tortillas, but the total fumonisins were reduced 50%. The total FB1 plus HFB1 in the residual lime water and water washes of the nixtamal accounted for 50% of the total FB1 in the uncooked maize. HFB1 and FB1 were present in a 1:1 mol/L ratio in the water washes of the nixtamal, the masa dough and the cooked tortillas, whereas the ratio of HFB1:FB1 in lime water after steeping was 21. Water washes contained 11% of the FB1 that was in the uncooked maize. The results show that the traditional method reduced the total fumonisins in tortillas and reduced the sphinganine elevation (a biomarker closely correlated with fumonisin toxicity) in cells treated with extracts of tortillas compared with cells treated with extracts of contaminated maize.

Effect of baking and frying on the in vivo toxicity to rats of cornmeal containing fumonisins.

Fumonisins are mycotoxins produced by Fusarium verticillioides (=F. moniliforme) and other Fusarium species. They are found in corn and corn-based foods. Cooking decreases fumonisin concentrations in food products under some conditions; however, little is known about how cooking effects biological activity. Baked cornbread, pan-fried corncakes, and deep-fried fritters were made from cornmeal that was spiked with 5% w/w F. verticillioides culture material (CM). The cooked materials and the uncooked CM-spiked cornmeal were fed to male rats (n = 5/group) for 2 weeks at high (20% w/w spiked cornmeal equivalents) or low (2% w/w spiked cornmeal equivalents) doses. A control group was fed a diet containing 20% w/w unspiked cornmeal. Toxic response to the uncooked CM-spiked cornmeal and the cooked products included decreased body weight gain (high-dose only), decreased kidney weight, and microscopic kidney and liver lesions of the type caused by fumonisins. Fumonisin concentration, as determined by HPLC analysis, in the 20% w/w pan-fried corncake diet [92.2 ppm of fumonisin B(1) (FB(1))] was slightly, but not statistically significantly, lower than those of the 20% w/w baked cornbread (132.2 ppm of FB(1)), deep-fried fritter (120.2 ppm of FB(1)) and CM-spiked cornmeal (130.5 of ppm FB(1)) diets. Therefore, baking and frying had no significant effect on the biological activity or concentration of fumonisins in these corn-based products, and the results provided no evidence for the formation of novel toxins or "hidden" fumonisins during cooking.

Leaching and binding of fumonisins in soil microcosms.

Fumonisin B(1) (FB(1)) is a water-soluble mycotoxin produced by Fusarium verticillioides. Our research objectives were to determine the leaching of FB(1) through soils and FB(1) binding in soil. Leachate columns were used to determine the movement of FB(1) through soil. FB(1)-contaminated corn screenings or water extracts containing FB(1) were placed on the surface of soil columns. In 100% sand columns, FB(1) leaching was only slightly retarded, whereas at 50%, 75%, and 100% Cecil sandy loam, approximately 60%, 50%, and 20% of the FB(1) was recovered in the column leachate, respectively. The FB(1) retained on the 100% Cecil sandy loam column was tightly bound. However, approximately 75% of the bound FB(1) was released with 5% formic acid and 5% formic acid/acetonitrile (1:1), indicating that the nature of the interaction was probably ionic. The results suggest that FB(1) is quite stable in soils and, while tightly bound, under certain environmental conditions could be released.

Paradoxical role of tumor necrosis factor alpha in fumonisin-induced hepatotoxicity in mice.

Tumor necrosis factor alpha (TNFalpha) is involved in fumonisin-induced hepatotoxic effects in mice. The hepatic response to fumonisin B(1) (FB(1)) was reduced in transgenic animals lacking either of the two TNFalpha receptors. In the present study, we hypothesized that the effect of a similar fumonisin treatment in animals lacking either TNFalpha or both TNFalpha receptors would be considerably less than their wild type (WT) counterparts. The FB(1)-induced increase in circulating liver enzymes was enhanced by deletion of TNFalpha or unchanged in mice lacking both TNFalpha receptors. These findings corresponded with the degree of toxicity as established by microscopic examination of liver. FB(1) induced the expression of TNFalpha in the liver of all strains, except the animals with a deleted TNFalpha gene. The FB(1)-mediated increases in liver sphingosine or sphinganine paralleled the hepatotoxic responses. It is apparent that the presence of TNFalpha is not necessary for FB(1)-induced hepatotoxicity in mice and a lack of the function of this cytokine may aggravate the hepatotoxic responses to fumonisins, perhaps by preventing repair mechanisms or by expression of other signaling molecules. These observations were in accordance with our previous finding where over-expression of TNFalpha also protected against FB(1)-mediated hepatotoxicity, and with the reported beneficial functions of low-level TNFalpha in tissue regeneration.

Temporal expression of fumonisin B(1)-induced tumor necrosis factor-alpha and interferon gamma in mice.

Fumonisin B(1) (FB(1)), a toxic metabolite of Fusarium verticillioides, is a carcinogen and causative agent of various animal diseases. Our previous studies indicated the involvement of tumor necrosis factor-alpha (TNF alpha) in FB(1)-induced toxic responses. To further investigate the time-course of TNF alpha production and signaling, mice (four/group) were treated subcutaneously (s.c.) or per os (p.o.) with either vehicle or 25 mg/kg of FB(1) as a single dose and sacrificed at 0, 2, 4, 8, 12 and 24 h after treatment. The TNF alpha expression was increased in liver and kidney after both routes of FB(1) exposure without any alterations in spleen. The p.o.-route FB(1) treatment caused greater hepatotoxicity compared to the s.c. route, as depicted by increased alanine aminotransferase and aspartate aminotransferase level in plasma, observed only after p.o. FB(1) treatment. The increase in enzymes at 8 h after p.o. treatment correlated with the highest TNF alpha expression, also noted at 8 h after p.o. treatment, thus further confirming the involvement of TNF alpha in FB(1) toxicity. The interferon (IFN)-gamma expression was increased in liver at 4 h after p.o. FB(1) treatment, suggesting a possible combined role of TNF alpha and IFN gamma in their induction and hepatotoxicity.