Alkaline cooking (nixtamalisation) and the reduction in the in vivo toxicity of fumonisin-contaminated corn in a rat feeding bioassay.

Nixtamalisation is a widely used food processing method in which whole kernel corn is cooked and steeped in alkaline water. It reduces the amount of fumonisin B1 (FB1) that can be detected after cooking. However, the fate of FB1 during nixtamalisation is not fully understood and potentially toxic reaction products, including matrix-associated "masked" FB1 forms that are not detected by routine analytical methods might remain in nixtamalised corn. To assess how nixtamalisation of whole kernel corn affects fumonisin toxicity, male rats were fed diets containing low, mid or high levels of uncooked (LU, MU, HU) or alkaline cooked (LC, MC, HC) FB1-contaminated corn for 3 weeks. The control diet contained uncontaminated corn only. Apoptotic kidney lesions of the type caused by FB1 were not found in the LC or MC groups. Lesions in the group fed HC were minimal and less severe than those found in the rats fed LU, MU or HU. Furthermore, significantly increased sphinganine and sphingosine concentrations indicative of FB1 exposure were found in the kidneys of the rats given LU, MU or HU. Concentrations were also elevated, but to a lesser extent, in rats fed HC, whereas sphinganine and sphingosine concentrations in rats given LC or MC did not differ from the control group. FB1 concentrations in the LC (0.08 mg kg(-1)), MC (0.13 mg kg(-1)) and HC (0.37 mg kg(-1)) diets were markedly reduced compared with their LU (1.8 mg kg(-1)), MU (3.5 mg kg(-1)) and HU (4.2 mg kg(-1)) counterparts as determined by HPLC (n = 1 analysis/diet). Taken together, the findings show that nixtamalisation is an effective cooking method for reducing the potential toxicity of FB1 contaminated corn.

The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides.

The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi.

Maternal fumonisin exposure as a risk factor for neural tube defects.

Fumonisins are mycotoxins produced by the fungus F. verticillioides, a common contaminant of maize (corn) worldwide. Maternal consumption of fumonisin B(1)-contaminated maize during early pregnancy has recently been associated with increased risk for neural tube defects (NTDs) in human populations that rely heavily on maize as a dietary staple. Experimental administration of purified fumonisin to mice early in gestation also results in an increased incidence of NTDs in exposed offspring. Fumonisin inhibits the enzyme ceramide synthase in de novo sphingolipid biosynthesis, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. Increased sphingoid base metabolites (i.e., sphinganine-1-phosphate) may perturb signaling cascades involved in embryonic morphogenesis by functioning as ligands for sphingosine-1-P (S1P) receptors, a family of G-protein-coupled receptors that regulate key biological processes such as cell survival/proliferation, differentiation and migration. Fumonisin-induced depletion of glycosphingolipids impairs expression and function of the GPI-anchored folate receptor (Folr1), which may also contribute to adverse pregnancy outcomes. NTDs appear to be multifactorial in origin, involving complex gene-nutrient-environment interactions. Vitamin supplements containing folic acid have been shown to reduce the occurrence of NTDs, and may help protect the developing fetus from environmental teratogens. Fumonisins appear to be an environmental risk factor for birth defects, although other aspects of maternal nutrition and genetics play interactive roles in determining pregnancy outcome. Minimizing exposures to mycotoxins through enhanced agricultural practices, identifying biomarkers of exposure, characterizing mechanisms of toxicity, and improving maternal nutrition are all important strategies for reducing the NTD burden in susceptible human populations.

Fumonisin concentrations and in vivo toxicity of nixtamalized Fusarium verticillioides culture material: evidence for fumonisin-matrix interactions.

The toxic potential of nixtamalized foods can be underestimated if, during cooking, reversible fumonisin-food matrix interactions reduce the amount of mycotoxin that is detected but not the amount that is bioavailable. Fusarium verticillioides culture material (CM) was nixtamalized as is (NCM) or after mixing with ground corn (NCMC). Additional portions were sham nixtamalized without (SCM) or with corn (SCMC). Nixtamalization and sham nixtamalization reduced FB(1); CM, NCM, and SCM diets contained 9.08, 2.08, and 1.19 ppm, respectively. FB(1) was further reduced in the NCMC (0.49 ppm) but not the SCMC (1.01 ppm) diets compared to their NCM and SCM counterparts. Equivalent weights of the cooked products, uncooked CM, corn (UC) or nixtamalized UC (NUC) were fed to rats for up to three weeks. Kidney lesions in the NCM-fed group were less severe than in the CM-fed, positive control group and no lesions were found in the NCMC and other groups. Group kidney sphinganine (biomarker of fumonisin exposure) concentrations decreased in the order: CM (absolute concentration (nmol/g)=600-800)>NCM (400-600)>SCM and SCMC (30-90)>NCMC, UC and NUC (<8). Together, these results suggest that mycotoxin-corn matrix interactions during nixtamalization reduce the bioavailability and toxicity of FB(1).

International shipping of fumonisins from maize extracts on C18 sorbent.

Fumonisins are mycotoxins found in maize. In developing countries, the resources required for analysis are often lacking, and the shipping of maize between countries can be difficult since the importation of plant materials requires permits/inspection to prevent the entry of pests that frequently infest maize. A simple, safe and legal method for shipping maize extracts to the USA was needed to conduct a survey of fumonisins in Central America. The objective was to develop a method for isolating and shipping maize extracts for fumonisin analysis so as to facilitate a survey of fumonisin exposure. The results indicate that fumonisins in acetonitrile:water extracts of maize can be isolated on C18 cartridges, held for at least 3 days at 22 degrees C and then an additional 4 days at 4 degrees C before elution and analysis with no losses. This method allows the importation and analysis of maize samples from foreign locations without complications from international safety concerns.

Fumonisins: current research trends in developmental toxicology.

Fumonisin B1 (FB1) is a mycotoxin produced byFusarium verticillioides that is found in maize and maize-based foods. Reproductive studies in CD1 mice, rats and rabbits initially found no evidence that fumonisins are teratogenic. However, more recent findings suggest that they might increase the risk of neural tube defects (NTDs) in populations consuming large amounts of fumonisin-contaminated corn. When ≥15 mg/kg body weight fumonisin B1 (FB1) was given to pregnant LM/Bc mice by intraperitoneal (ip) injection, all litters were positive for NTDs. To determine if NTD induction is unique to the inbred LM/Bc mouse strain, NTD induction in LM/Bc and CD1 mice was compared: (a) in a study in whichF. verticillioides culture material providing ≤150 ppm FB1 was fed to female mice before and during gestation, and (b) in a study in which FB1 was given by ip injection to CD1 dams on gestation days 7 and 8, the critical time for NTD development. In the feeding study, one of five LM/Bc litters from dams fed the 150 ppm FB1 diet was positive for NTDs whereas no NTDs were found in the CD1 litters. In the ip injection study, 40% of the litters at the highest dose tested, 45 mg/kg body weight, were positive for NTDs and one of nine low-dose (15 mg/kg body weight) litters was also positive. Thus, FB1 induced NTDs in both LM/Bc and CD1 mice although the latter strain appears less sensitive. Comparative investigations using these strains will be useful for elucidating the mechanisms underlying fumonisin-induced NTDs in mice and determining the suitability of mouse models for studying the relationships between fumonisins and NTDs in humans.

Activated carbon does not prevent the toxicity of culture material containing fumonisin B1 when fed to weanling piglets.

Fumonisins are mycotoxins found primarily in corn and corn products that are produced by Fusarium verticillioides, F. proliferatum, and several other Fusarium species. The toxicity of fumonisin B1 (FB) from culture material with and without activated carbon was evaluated using weanling piglets. Fifty-six weanling pigs were assigned to one of four treatments diets based on BW. The treatment diets were 1) control = corn-soybean basal diet with < 2 ppm FB; 2) AC = control + activated carbon at 1% of the diet, as fed; 3) FB = control + culture material (formulated to contain 30 ppm FB, as-fed basis); and 4) AC + FB = control + activated carbon at 1% of the diet as fed + culture material (formulated to contain 30 ppm FB). A total of four replicates of four pigs per pen for the control and AC treatments and three piglets per pen for the FB and AC + FB treatments were used. Feed and water were offered ad libitum for the duration of the 42-d experiment. Compared with pigs fed the control or AC diets, pigs receiving the two FB-contaminated diets (FB or AC + FB) had lower G:F (P < 0.01), higher serum enzyme activities of gamma-glutamyltransferase and glutamic oxaloacetic transaminase (P < 0.05), and higher concentrations of cholesterol, free sphinganine, sphingosine-1-phosphate, and sphinganine 1-phosphate (P < 0.05). Although animals consuming FB diets showed no signs of respiratory distress, all pigs consuming either the FB or the AC + FB diets had marked pulmonary edema. Lesions were observed in the lungs, heart, and liver of pigs fed the FB or AC + FB diets, and treatment-associated changes also were seen in the pancreas, intestines, spleen, and lymph nodes. No lesions were observed in the brain. In liver, lung, heart, pancreas, spleen, intestines, and lymph nodes, the histopathological effects observed were more severe in the AC + FB group, suggesting that the AC treatment worsened the toxic effects of FB. Additionally, immunological measurements of macrophage function (CD14) were affected (P < 0.05) by the consumption of the FB diets. The consumption of FB diets containing 30 ppm fumonisin B1 from cultured material significantly affected performance, biochemical measurements, and organ pathology in weanling pigs. The addition of activated carbon at the rate of 1% to the diet was not effective in protecting against the detrimental effects of fumonisin consumption.

Host resistance to Trypanosoma cruzi infection is enhanced in mice fed Fusarium verticillioides (=F. moniliforme) culture material containing fumonisins.

Fumonisins, metabolites of Fusarium verticillioides (=F. moniliforme) and related fungi that occur naturally on corn, elicit various organ- and species-specific toxicities. However, immunologic effects of fumonisins are not well characterized. BALB/c mice were fed diets containing F. verticillioides culture material (CM) providing 50 (LD) or 150 (HD) ppm fumonisins (FB(1)+FB(2)) beginning 1 week before and continuing 5 weeks after challenge with the myotropic Brazil strain of T. cruzi. A control group (ZD) was fed a diet lacking CM. The LD and HD diets caused increases in tissue sphinganine/sphingosine ratios and minimum to mild hepatotoxicity, both of which are typically induced by fumonisins. Nitric oxide (NO) production by peritoneal macrophages from HD mice was significantly higher than by peritoneal macrophages from ZD mice on day 14 after challenge. NO production also was stimulated in macrophages from ZD mice, but the peak response did not occur until day 26 after challenge. Compared with ZD mice, LD and HD mice exhibited reduced parasitemia and decreased numbers of pseudocysts in cardiac muscle. Thus, the CM increased host resistance to T. cruzi by accelerating NO production by macrophages or otherwise enhancing the immune response. The findings provide additional evidence that fumonisins modulate immune function.

Persistence and reversibility of the elevation in free sphingoid bases induced by fumonisin inhibition of ceramide synthase.

These studies determined (1) the time course for sphingoid base elevation in the small intestines, liver, and kidney of mice following a single 25 mg/kg body weight (bw) oral dose (high dose) of fumonisin B(1) (FB(1)), (2) the minimum threshold dose of FB(1) that would prolong the elevated sphingoid base concentration in kidney following the single high dose, and (3) the importance of the balance between the rate of sphingoid base biosynthesis and degradation in the persistence of sphingoid base accumulation. Following the high dose of FB(1), there was an increase in sphinganine in intestinal cells and liver that peaked at 4 to 12 h and declined to near the control level by 48 h. In kidney, sphinganine peaked at 6-12 h but remained elevated until 72 h, approaching control levels at 96-120 h. Oral administration of 0.03 mg FB(1)/kg bw (low dose) for 5 days had no effect on the sphingoid bases in kidney. However, following an initial high dose, daily administration of the low dose prolonged the elevation in kidney sphinganine compared to mice receiving a single high dose. Thus, a single exposure to a high dose of FB(1) followed by daily exposure at low levels will prolong the elevation of sphinganine in kidney. In cultured renal cells FB(1) was rapidly eliminated, but elevated sphinganine was persistent. This persistence in renal cells was rapidly reversed in the presence of the serine palmitoyltransferase inhibitor (ISP-1), indicating that the persistence was due to differences in the rates of sphinganine biosynthesis and degradation. The in vivo persistence in kidney may be due to similar differences.

Barriers and resistance to informatics in behavioral health.

The levels of resistance to a new informatics system can vary widely both between and among specific groups. The relevance to today's behavioral medicine area is obvious. The aim of effective change management techniques is not to eliminate all resistance. This is typically impossible when a group of any size is involved. The aims are (1) to keep initial general resistance at reasonable levels, (2) to pre-vent that initial resistance from growing to serious levels, and (3) to identify and deal with any pockets of serious resistance that do occur despite the previous efforts. His article outlines areas of resistance to behavioral informatics and offers suggestions for overcoming the resistance.

Instability of N-acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides. It inhibits ceramide synthase, which is a proposed underlying mechanism responsible for the myriad of toxic endpoints observed. We previously reported that N-acetylation of FB1 prevents ceramide synthase inhibition, but cautioned that impure preparations of FA1 can contain a contaminant with the ability to inhibit ceramide synthase. We now report that FA1 spontaneously rearranges to O-acetylated analogs. These rearrangement products are putative inhibitors of ceramide synthase. Rat liver slices exposed to impure FA1 containing O-acetylated FB1 had sphinganine/sphingosine (Sa:So) ratios of 1.15-1.64. Control slices had Sa:So ratios of 0.07-0.24. Clean-up to remove the O-acetylated FB1 yielded purified FA1, which produced Sa:So ratios in liver slices of 0.08-0.18. After storage for approximately 1 year as either a dry powder in a desiccator, or as a dried film at 4 degrees C, the purified FA1 again contained O-acetylated FB1, and was capable of ceramide synthase inhibition. FA1 was most stable in neutral solution, but in acidic solution the equilibrium shifted towards the O-acetylated forms. FA1 in solid form also rearranged, but more slowly than in acid solution. As FA1 is considerably less cytotoxic than FB1, these results provide additional support for the conclusion that a primary amino group is necessary for both ceramide synthase inhibition and toxicity.

Fumonisin-induced tumor necrosis factor-alpha expression in a porcine kidney cell line is independent of sphingoid base accumulation induced by ceramide synthase inhibition.

Previous studies have shown that fumonisin B1 (FB1) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-alpha (TNFalpha) plays an important role in FB1 toxicity and the expression of TNFalpha mRNA in liver and kidney is increased following FB1 exposure in mice. The objective of the current study was to investigate whether these two events (sphingoid bases accumulation and TNFalpha induction) are dependent on each other. An increase in expression of TNFalpha mRNA was detected in LLC-PK1 cells as early as 4 h after FB1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNFalpha mRNA and FB1 concentration. Increases of intracellular sphingoid bases were also detected after 4 h of FB1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNFalpha. Inhibition of sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free sphingoid bases in response to FB1, but it did not prevent the induction of TNFalpha expression. Results indicate that FB1-induced increase in TNFalpha expression is independent of sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK1 cells.

An overview of rodent toxicities: liver and kidney effects of fumonisins and Fusarium moniliforme.

Fumonisins are produced by Fusarium moniliforme F. verticillioides) and other Fusarium that grow on corn worldwide. They cause fatal toxicoses of horses and swine. Their effects in humans are unclear, but epidemiologic evidence suggests that consumption of fumonisin-contaminated corn contributes to human esophageal cancer in southern Africa and China. Much has been learned from rodent studies about fumonisin B1(FB1), the most common homologue. FB1 is poorly absorbed and rapidly eliminated in feces. Minor amounts are retained in liver and kidneys. Unlike other mycotoxins, fumonisins cause the same liver cancer promotion and subchronic (studies (3/4) 90 days) liver and kidney effects as (italic)F. moniliforme. FB 1 induces apoptosis of hepatocytes and of proximal tubule epithelial cells. More advanced lesions in both organs are characterized by simultaneous cell loss (apoptosis and necrosis) and proliferation (mitosis). Microscopic and other findings suggest that an imbalance between cell loss and replacement develops, a condition favorable for carcinogenesis. On the molecular level, fumonisins inhibit ceramide synthase, and disrupt sphingolipid metabolism and, theoretically, sphingolipid-mediated regulatory processes that influence apoptosis and mitosis. Liver sphingolipid effects and toxicity are correlated, and ceramide synthase inhibition occurs in liver and kidney at doses below their respective no-observed-effect levels. FB1 does not cross the placenta and is not teratogenic in vivoin rats, mice, or rabbits, but is embryotoxic at high, maternally toxic doses. These data have contributed to preliminary risk evaluation and to protocol development for carcinogenicity and chronic toxicity studies of FB1 in rats and mice.

Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins.

Sphingolipids have important roles in membrane and lipoprotein structure and in cell regulation as second messengers for growth factors, differentiation factors, cytokines, and a growing list of agonists. Bioactive sphingolipids are formed both by the turnover of complex sphingolipids and as intermediates of sphingolipid biosynthesis. Usually, the amounts are highly regulated; however, by inhibiting ceramide synthase, fumonisins block the biosynthesis of complex sphingolipids and cause sphinganine (and sometimes sphingosine) to accumulate. Where the mechanism has been studied most thoroughly, the accumulation of sphingoid bases is a primary cause of the toxicity of fumonisin B (FB). Nonetheless, the full effects of fumonisins probably involve many biochemical events. The elevations in sphingoid bases also affect the amounts of other lipids, including the 1-phosphates and N-acetyl derivatives of sphinganine. Furthermore, the aminopentol backbone of FB1 (AP1) is both an inhibitor and a substrate for ceramide synthase, and the resultant N-palmitoyl-AP1 (PAP1) is an even more potent inhibitor of ceramide synthase (presumably as a product analog). PAP1 is 10 times more toxic than FB1 or AP1 for HT-29 cells in culture, and hence may play a role in the toxicity of nixtamalized fumonisins. All these processes--the effects of fumonisins on sphingolipid metabolism, the pathways altered by perturbation of sphingolipid metabolism, and the complex cellular behaviors regulated by sphingolipids--must be borne in mind when evaluating the pathologic effects of fumonisins.

Sphingolipid perturbations as mechanisms for fumonisin carcinogenesis.

There is a great deal of evidence that altered sphingolipid metabolism is associated with fumonisin-induced animal diseases including increased apoptotic and oncotic necrosis, and carcinogenesis in rodent liver and kidney. The biochemical consequences of fumonisin disruption of sphingolipid metabolism most likely to alter cell regulation are increased free sphingoid bases and their 1-phosphates, alterations in complex sphingolipids, and decreased ceramide (CER) biosynthesis. Because free sphingoid bases and CER can induce cell death, the fumonisin inhibition of CER synthase can inhibit cell death induced by CER but promote free sphingoid base-induced cell death. Theoretically, at any time the balance between the intracellular concentration of effectors that protect cells from apoptosis (decreased CER, increased sphingosine 1-phosphate) and those that induce apoptosis (increased CER, free sphingoid bases, altered fatty acids) will determine the cellular response. Because the balance between the rates of apoptosis and proliferation is important in tumorigenesis, cells sensitive to the proliferative effect of decreased CER and increased sphingosine 1-phosphate may be selected to survive and proliferate when free sphingoid base concentration is not growth inhibitory. Conversely, when the increase in free sphingoid bases exceeds a cell's ability to convert sphinganine/sphingosine to dihydroceramide/CER or their sphingoid base 1-phosphate, then free sphingoid bases will accumulate. In this case cells that are sensitive to sphingoid base-induced growth arrest will die and insensitive cells will survive. If the cells selected to die are normal phenotypes and the cells selected to survive are abnormal, then cancer risk will increase.

Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisin B(1) as an example.

The rates of cell proliferation and cell loss in conjunction with the differentiation status of a tissue are among the many factors contributing to carcinogenesis. Nongenotoxic (non-DNA reactive) chemicals may affect this balance by increasing proliferation through direct mitogenesis or through a regenerative response following loss of cells through cytotoxic (oncotic) or apoptotic necrosis. In a recent NTP study in Fischer rats and B6C3F(1) mice, the mycotoxin fumonisin B(1) caused renal carcinomas in male rats and liver cancer in female mice. In an earlier study in male BD-IX rats, fumonisin B(1) caused hepatic toxicity and hepatocellular carcinomas. An early effect of fumonisin B(1) exposure in these target organs is apoptosis. However, there is also some evidence of oncotic necrosis following fumonisin B(1) administration, especially in the liver. Induction of apoptosis may be a consequence of ceramide synthase inhibition and disruption of sphingolipid metabolism by fumonisin B(1). Fumonisin B(1) is not genotoxic in bacterial mutagenesis screens or in the rat liver unscheduled DNA-synthesis assay. Fumonisin B(1) may be the first example of an apparently nongenotoxic (non-DNA reactive) agent producing tumors through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.

Fumonisin B1 promotes aflatoxin B1 and N-methyl-N'-nitro-nitrosoguanidine-initiated liver tumors in rainbow trout.

Laboratory studies have described the carcinogenicity of fumonisin B1 (FB1) in rodents and epidemiological evidence suggests an association between FB1 (a mycotoxin produced by Fusarium moniliforme) and cancer in humans. This study was designed to reveal in rainbow trout, a species with very low spontaneous tumor incidence, if FB1 was (i) a complete carcinogen, in the absence of an initiator; (ii) a promoter of liver tumors in fish initiated as fry with aflatoxin B1 (AFB1); and (iii) a promoter of liver, kidney, stomach, or swim bladder tumors in fish initiated as fry with N-methyl-N'-nitro-nitrosoguanidine (MNNG). FB1 was not a complete carcinogen in trout. No tumors were observed in any tissue of fish fed diets containing 0, 3.2, 23, or 104 ppm FB1 for a total of 34 weeks (4 weeks FB1 exposure, 2 weeks outgrowth on control diet, followed by 30 weeks FB1 diet) in the absence of a known initiator. FB1 promoted AFB1 initiated liver tumors in fish fed > or = 23 ppm FB1 for 42 weeks. A 1-week pretreatment of FB1 did not alter the amount of liver [3H]AFB1 DNA adducts, which suggests that short-term exposure to FB1 will not alter phase I or phase II metabolism of AFB1. In MNNG-initiated fish, liver tumors were promoted in the 104 ppm FB1 treatment (42 weeks), but FB1 did not promote tumors in any other tissue. Tumor incidence decreased in kidney and stomach in the 104 ppm FB1 treatment of MNNG-initiated trout. The FB1 promotional activity in AFB1-initiated fish was correlated with disruption of sphingolipid metabolism, suggesting that alterations in associated sphingolipid signaling pathways are potentially responsible for the promotional activity of FB1 in AFB1-initiated fish.

Decreased fumonisin hepatotoxicity in mice with a targeted deletion of tumor necrosis factor receptor 1.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides and related fungi infests corn and other cereals, and causes a variety of toxic effects in different mammalian species. Hepatotoxicity is a common toxic response in most species. The cellular responses of FB1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a corresponding induction of tumor necrosis factor alpha (TNFalpha). We recently reported that FB1 hepatotoxicity was considerably reduced in a mouse strain lacking tumor necrosis factor receptor 2 (TNFR2 or TNFR1b). To further investigate the relative contribution of the two TNFalpha receptors (TNFR1 and TNFR2 or P55 and P75 receptors) we evaluated the hepatotoxicity of FB1 in male C57BL/6J mice (WT) and a corresponding TNFR1 knockout (TNFRKO) strain, genetically modified by a targeted deletion of this receptor. The hepatotoxic effects of five daily injections of 2.25 mg/kg per day of FB1 were observed in WT but were reduced in TNFRKO, evidenced by the microscopic evaluation of the liver and increased concentrations of circulating alanine aminotransferase and aspartate aminotransferase. FB1 induced the expression of TNFalpha, and similar increases in free sphinganine and sphingosine in livers of both WT and TNFRKO mice. Results indicated that both P55 and P75 receptors are required for FB1-induced hepatotoxicity and TNFalpha plays an important role in such response in mouse liver.

Disruption of sphingolipid metabolism in small intestines, liver and kidney of mice dosed subcutaneously with fumonisin B(1).

Fumonisin B(1) is a fungal inhibitor of ceramide synthase, a key enzyme in the de novo biosynthesis of sphingolipids. The resulting increase in tissue free sphinganine (and sometimes sphingosine) is used as a biomarker for fumonisin exposure. This study determined whether a single subcutaneous injection of fumonisin B(1) could cause an increase in free sphingoid bases in the intestinal epithelial cells of mice over 24 hr. It was hypothesized that fumonisin administered subcutaneously would be excreted into the small intestine via biliary excretion, and this should be detectable by increased sphingoid bases in the intestine. A significant time-dependent increase in sphingoid bases occurred in the intestine and liver peaking at 4-8 hr and declining to control levels by 24 hr. In the kidney the increase in free sphinganine was persistent. The parallel time course of the change in sphinganine in the intestine and liver suggested fumonisin B(1) was rapidly excreted into the small intestine. Rapid cell turnover in the intestine could account for the reversal of the sphinganine increase. The rapid return to the control level in liver was unexpected since ceramide synthase inhibition in cultured cells is persistent suggesting that liver handles fumonisin B(1) or sphingoid bases quite differently than kidney.

Fumonisin hepatotoxicity is reduced in mice carrying the human tumour necrosis factor alpha transgene.

Our previous studies have indicated that tumour necrosis factor alpha (TNFalpha) is involved in fumonisin B1 (FB1)-induced toxic responses. To investigate the role of TNFalpha in FB1 toxicity further we employed male transgenic mice expressing human TNFalpha gene (TG) and their wild-type equivalent C57BL/6 (WT). It was hypothesized that TG animals would have enhanced response to FB1. Repeated subcutaneous treatment of animals with 2.25 mg/kg per day of FB1 for 5 days caused minimal changes in body weight, organ weights, blood cell counts, and TNFalpha levels in plasma 1 day after the last injection. The mRNA for TNFalpha in liver increased in both TG and WT after FB1 treatment, providing evidence that FB1 induces hepatic TNFalpha expression. Liver and kidney lesions were found in TG after FB1 treatment; however, liver lesions seen in FB1-treated TG were considerably less than those observed in WT. The decreased hepatotoxicity in TG after FB1 treatment correlated with plasma concentrations of alanine aminotransferase and aspartate aminotransferase. Free sphinganine levels increased significantly in both the liver and kidney of WT and TG mice treated with FB1. The increase of free sphinganine in the liver from TG mice was 40% less than in WT mice and paralleled the changes in serum liver enzymes. Regional brain neurotransmitters and their metabolites were increased to a similar extent by FB1 in both WT and TG mice. Since the data did not support the original hypothesis, we investigated the levels of NFkappaB in liver. The cytosolic NFkappaB was significantly higher in TG compared with WT. Induction of NFkappaB, caused by increased endogenous production of TNFalpha, is a possible explanation of decreased FB1 hepatotoxicity in TG. The results suggest a protective role for NFkappaB in FB1-induced liver damage.