Mycotoxins were not associated with environmental enteropathy in a cohort of Tanzanian children.

Enteropathy is a pathophysiological condition characterized by decreased intestinal barrier function and absorption. Past studies have hypothesized that mycotoxins might impair children's growth by causing intestinal enteropathy, including interactions between mycotoxins and pathogens. We investigated the association of two mycotoxins, aflatoxin B1 (AFB1 ) and fumonisin B1 (FB1 ), independently and in conjunction with microbial pathogens, with fecal biomarkers of environmental enteropathy in children. As part of a larger MAL-ED study, 196 children were recruited in Haydom, Tanzania, and followed for the first 36 months of life. The gut inflammation biomarkers myeloperoxidase (MPO), neopterin (NEO), and alpha-1-antitrypsin (A1AT) were analyzed in stool samples at 24 months; with mean concentrations 5332.5 ng/L MPO, 807.2 nmol/L NEO, and 0.18 mg/g A1AT. Forty-eight children were measured for AFB1 -lys, with a mean of 5.30 (95% CI: 3.93-6.66) pg/mg albumin; and 87 were measured for FB1 , with a mean of 1.25 (95% CI: 0.72-1.76) ng/ml urine. Although the pathogens adenovirus and Campylobacter were associated with A1AT (p = 0.049) and NEO (p = 0.004), respectively, no association was observed between aflatoxin (MPO, p = 0.30; NEO, p = 0.08; A1AT, p = 0.24) or fumonisin (MPO, p = 0.38; NEO, p = 0.65; A1AT, p = 0.20) exposure and any gut inflammation biomarkers; nor were interactive effects found between mycotoxins and pathogens in contributing to intestinal enteropathy in this cohort. Although further studies are needed to confirm these results, it is possible that mycotoxins contribute to child growth impairment via mechanisms other than disrupting children's intestinal function.

Ceramide synthase inhibition by fumonisins: a perfect storm of perturbed sphingolipid metabolism, signaling, and disease.

Fumonisins are mycotoxins that cause diseases of plants and, when consumed by animals, can damage liver, kidney, lung, brain, and other organs, alter immune function, and cause developmental defects and cancer. They structurally resemble sphingolipids (SLs), and studies nearly 30 years ago discovered that the most prevalent fumonisin [fumonisin B1 (FB1)] potently inhibits ceramide synthases (CerSs), enzymes that use fatty acyl-CoAs to N-acylate sphinganine (Sa), sphingosine (So), and other sphingoid bases. CerS inhibition by FB1 triggers a "perfect storm" of perturbations in structural and signaling SLs that include: reduced formation of dihydroceramides, ceramides, and complex SLs; elevated Sa and So and their 1-phosphates, novel 1-deoxy-sphingoid bases; and alteration of additional lipid metabolites from interrelated pathways. Moreover, because the initial enzyme of sphingoid base biosynthesis remains active (sometimes with increased activity), the impact is multiplied by the continued production of damaging metabolites. Evidence from many studies, including characterization of knockout mice for specific CerSs and analyses of human blood (which found that FB1 intake is associated with elevated Sa 1-phosphate), has consistently pointed to CerS as the proximate target of FB1 It is also apparent that the changes in multiple bioactive lipids and related biologic processes account for the ensuing spectrum of animal and plant disease. Thus, the diseases caused by fumonisins can be categorized as "sphingolipidoses" (in these cases, due to defective SL biosynthesis), and the lessons learned about the consequences of CerS inhibition should be borne in mind when contemplating other naturally occurring and synthetic compounds (and genetic manipulations) that interfere with SL metabolism.

Dietary Fumonisin and Growth Impairment in Children and Animals: A Review.

Fumonisins are mycotoxins produced primarily by the fungi Fusarium verticillioides and F. proliferatum, which colonize maize in tropical, subtropical, and temperate climates worldwide. Fumonisin exposure is the highest in rural populations that consume large amounts of maize and maize products. Among them, infants and young children are the most vulnerable when they are weaned onto maize-based foods. Therefore, it is critical to understand the impact of fumonisin on children's health and growth. This review describes the evidence linking fumonisin exposure to child growth impairment. First, toxicological studies that attempt to elucidate the mechanism by which fumonisin may impair growth are discussed. Next, a description is given of candidate biomarkers for accurately capturing fumonisin exposure in humans. Potential human health effects of fumonisin exposure beyond growth impairment, including esophageal cancer and neural tube defects (NTDs), are briefly described. We review epidemiological studies that collectively show an increasing weight of evidence linking fumonisin exposure to growth impairment, particularly in world regions where young children consume maize-based weaning foods. Additionally, the evidence linking fumonisin exposure to growth impairment in plants and a variety of animal species are reviewed. Finally, we describe interventions to reduce fumonisin in parts of the world where dietary fumonisin exposure is high and is likely to predispose populations to increased health risk.

Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts.

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs.

Increased sphingoid base-1-phosphates and failure of neural tube closure after exposure to fumonisin or FTY720.

BACKGROUND: Fumonisin B(1) (FB(1)) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB(1)-contaminated food is associated with increased risk for neural tube defects (NTDs). FB(1) induces NTDs in inbred LM/Bc mice. FB(1) inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. METHODS: Pregnant SWV and LM/Bc mice were treated with FB(1) (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5-8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5-8.5). LC/MS was used to detect sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB(1) (40 µM for 24 hr) and LC/MS was used to detect sphingoid base-1-phosphates. RESULTS: FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB(1) or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB(1), and Sa1P was higher in MEFs generated from the FB(1)-NTD-susceptible LM/Bc strain. CONCLUSIONS: Elevated sphingoid base-1-P after FB(1) or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB(1) or FTY720. Sa1P may represent a biomarker for FB(1)-NTD risk assessment.

Dietary and socioeconomic risk factors for fumonisin exposure among women of reproductive age in 18 municipalities in Guatemala from 2013 to 2014.

Fumonisin exposure is common in populations where maize is a dietary staple, such as in Guatemala, and has been associated with negative health outcomes including neural tube defects. The objective of this study was to estimate fumonisin B1 (FB1) exposure among Guatemalan reproductive-age women and develop a better understanding of the dietary and sociodemographic risk factors for exposure. A cross-sectional study in 18 municipalities in Guatemala was conducted. Midwives and study nurses enrolled consenting women and collected individual and household demographic and socioeconomic data. A food frequency questionnaire was administered to estimate quantity and types of food products consumed. A urine sample was collected and urinary fumonisin B1 (uFB1) concentration was measured. A univariable analysis was conducted to identify predictors of low/high uFB1. Multivariable logistic regression was used to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). In total, 775 women had analyzable urine samples. Higher uFB1 levels were associated with speaking Mayan (OR = 2.33, 95% CI:1.44-3.77), less than high school education (OR = 1.61, 95% CI:1.12-2.30), increasing dietary proportion of maize-based foods (OR = 1.02, 95% CI:1.01-1.03), and consumption of tostadas (fried tortillas) (OR = 1.11, 95% CI:1.02-1.22). Lower uFB1 levels were associated with consumption of highly processed maize-based foods (OR = 0.93, 95% CI:0.87-0.99). Tortillas were the most frequently consumed maize-based food among study participants and significantly associated with high uFB1 exposure in the univariable but not multivariable analysis. Consumption of >4,750 grams/week of maize-based foods, >5,184 g/week of locally produced maize-based foods, and >110 servings/week of tortillas were also significantly associated with high uFB1 exposure in univariable analysis. Populations with low socioeconomic status/education levels and high consumption of maize-based foods had higher fumonisin exposure. Interventions aimed at reducing the risk of exposure to mycotoxins through maize in Guatemala, including the increased consumption of non-maize-based foods, should be further explored.

Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings.

The filamentous ascomycete Fusarium verticillioides is a pathogen of maize and produces the fumonisin mycotoxins. However, a distinct population of F. verticillioides is pathogenic on banana and does not produce fumonisins. Fumonisin-producing strains from maize cause leaf lesions, developmental abnormalities, stunting, and sometimes death of maize seedlings, whereas fumonisin-nonproducing banana strains do not. A Southern analysis of banana strains did not detect genes in the fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. Nucleotide sequence analysis of the genomic region carrying the flanking genes revealed that the FUM cluster was absent in banana strains except for portions of FUM21 and FUM19, which are the terminal genes at each end of the cluster. Polymerase chain reaction analysis confirmed the absence of the cluster in all banana strains examined. Cotransformation of a banana strain with two overlapping cosmids, which together contain the entire FUM cluster, yielded fumonisin-producing transformants that were pathogenic on maize seedlings. Conversely, maize strains that possess the FUM cluster but do not produce fumonisins because of mutations in FUM1, a polyketide synthase gene, were not pathogenic on maize seedlings. Together, the data indicate that fumonisin production may have been lost by deletion of the FUM cluster in the banana population of F. verticillioides but that fumonisin production could be restored by molecular genetic complementation. The results also indicate that fumonisin production by F. verticillioides is required for development of foliar disease symptoms on maize seedlings.

A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings.

The fungus Fusarium verticillioides is a pathogen of many plants and produces fumonisins. In addition to their well-studied animal toxicoses, these toxins contribute to the development of maize seedling disease in susceptible maize varieties. Fumonisin disruption of sphingolipid biosynthesis occurs during pathogenesis. An extraction method was developed for the simultaneous analysis of fumonisins B(1) (FB(1)), B(2) (FB(2)) and B(3) (FB(3)), free sphingoid bases and sphingoid base 1-phosphates in maize tissues by liquid chromatography/linear ion trap tandem mass spectrometry. The method involved a single extraction using 1:1 acetonitrile:water + 5% formic acid (1 ml per 10 mg tissue). Mean recoveries ranged from approximately 50 to 99 percent, and limits of detection ranged from 10 fg microl(-1) to 6900 fg microl(-1). To test the efficacy of the method, seeds of a susceptible maize line were inoculated with a pathogenic, fumonisin-producing strain of F. verticillioides. The seedlings were then harvested, and fumonisin content, as well as sphingoid bases and their 1-phosphates, were measured in the leaf and root tissues. Fumonisin accumulation was significantly greater in leaf one compared to leaves two and three. While FB(1), FB(2), and FB(3) were detected in root tissues, FB(1) was preferentially accumulated in leaf tissues. Accumulation of sphingoid bases and their 1-phosphates was evident in roots and leaves of seedlings grown from inoculated seed, with the level of accumulation being similar in leaves 1, 2 and 3. The method developed was effective, fast, and sensitive for use in simultaneously measuring fumonisin in tissues and their effects on sphingolipid metabolite biomarkers of disease. The method should be useful for screening maize cultivars for susceptibility to F. verticillioides-induced seedling diseases.

Fumonisin mycotoxin contamination of corn-based foods consumed by potentially pregnant women in southern California.

OBJECTIVE: To evaluate a sample of locally available corn-based foods for fumonisin contamination. STUDY DESIGN: We analyzed 38 corn tortilla and masa flour samples from Los Angeles, San Diego and Tijuana, Mexico,for fumonisin contamination. Retail sources were diverse and not limited to Hispanic neighborhoods. RESULTS: Fumonisins were found in all samples. The median fumonisin B1 mycotoxin level was 84 ng/g, with a range of 1-729 (n = 38). The median total fumonisin level was 231 ng/g, with a range of 2.8-1,863. Levels of fumonisins differed by geographic site. CONCLUSION: Fumonisin contamination of corn-based foods in southern California is common. At levels of contamination > 1,000 ng/g, a 60-kg potentially pregnant woman could exceed the World Health Organization recommendations by eating 120 g (dry weight) of corn products daily. Fumonisin contamination may constitute a preventable risk for NTDs among susceptible reproductive-age women and their progeny.

Modulation of pre-neoplastic biomarkers induced by sequential aflatoxin B1 and fumonisin B1 exposure in F344 rats treated with UPSN clay.

Populations consuming aflatoxin (AF) and fumonisin (FN)-contaminated foods may be at increased risk for hepatocellular carcinoma (HCC) and developmental disorders; consequently, development of intervention strategies to reduce AF/FN-induced liver disease and adverse health effects in humans could be very useful. Calcium montmorillonite clay (NovaSil) has been shown to absorb AF in vitro, in multiple animal models, as well as in human studies. In the present study, we aimed to evaluate whether uniform particle size NovaSil (UPSN) possessed an ability to modulate the co-carcinogenic potentials of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in F344 rats. Sequential treatment of FB1 following AFB1 synergistically induces preneoplastic alterations as well as liver damage, indicating that AFB1 acts as an initiator while FB1 as a promoter in the carcinogenesis model, confirming findings from previous studies. The enterosorbent agent UPSN clay at dose of up to 0.5% in diet was shown to be effective in modulating the toxicity and carcinogenicity of co-exposure to AFB1 and FB1, as demonstrated by significant reduction in number and size of hepatic GST-P+ foci, in alterations indicative of liver toxicity, and in levels of AFB1 and FB1 biomarkers.

Exposure to aflatoxin and fumonisin in children at risk for growth impairment in rural Tanzania.

Growth impairment is a major public health issue for children in Tanzania. The question remains as to whether dietary mycotoxins play a role in compromising children's growth. We examined children's exposures to dietary aflatoxin and fumonisin and potential impacts on growth in 114 children under 36 months of age in Haydom, Tanzania. Plasma samples collected from the children at 24 months of age (N = 60) were analyzed for aflatoxin B1-lysine (AFB1-lys) adducts, and urine samples collected between 24 and 36 months of age (N = 94) were analyzed for urinary fumonisin B1 (UFB1). Anthropometric, socioeconomic, and nutritional parameters were measured and growth parameter z-scores were calculated for each child. Seventy-two percent of the children had detectable levels of AFB1-lys, with a mean level of 5.1 (95% CI: 3.5, 6.6) pg/mg albumin; and 80% had detectable levels of UFB1, with a mean of 1.3 (95% CI: 0.8, 1.8) ng/ml. This cohort had a 75% stunting rate [height-for-age z-scores (HAZ) < -2] for children at 36 months. No associations were found between aflatoxin exposures and growth impairment as measured by stunting, underweight [weight-for-age z-scores (WAZ) < -2], or wasting [weight-for-height z-scores (WHZ) < -2]. However, fumonisin exposure was negatively associated with underweight (with non-detectable samples included, p = 0.0285; non-detectable samples excluded, p = 0.005) in this cohort of children. Relatively low aflatoxin exposure at 24 months was not linked with growth impairment, while fumonisin exposure at 24-36 months based on the UFB1 biomarkers may contribute to the high growth impairment rate among children of Haydom, Tanzania; which may be associated with their breast feeding and weaning practices.

Estimated fumonisin exposure in Guatemala is greatest in consumers of lowland maize.

Fumonisin mycotoxins contaminate maize worldwide. Analysis of maize samples (n = 396) collected from fields in Guatemala from 2000 to 2003 found that lowland maize (<360 m) had significantly more fumonisin B1 than highland maize (>1200 m). For example, 78% of the lowland samples collected at harvest in 2002 contained >0.3 microg/g of fumonisin B1, whereas only 2% of the highland samples contained >0.3 microg/g. Maize from the 2002 crop collected from storage in the highlands just before the 2003 harvest contained significantly more fumonisin B1 compared with levels at harvest in 2002. All Fusarium-infected kernels analyzed from 9 random lowland locations in 2001 were infected with fumonisin-producing Fusarium verticillioides and no other Fusarium species, whereas in samples from the highlands, only 5% of the Fusarium-positive kernels were F. verticillioides. In 2005, maize samples (n = 236) from the 2004 crop were collected from local markets in 20 Departments across Guatemala. The analysis showed that maize from lowland locations was often highly contaminated with fumonisin and was frequently transported to and sold in highland markets. Thus, fumonisin exposure in the highlands will be greatest in groups that obtain their maize in the market place from commercial vendors. Based on a recall study and published consumption data, a preliminary assessment of daily intake of total fumonisins was estimated. Consumption of nixtamalized maize products made from >50% of the maize from commercial vendors in 2005 could result in exposure exceeding the recommended WHO provisional maximal tolerable daily intake.

Inducible nitric oxide has protective effect on fumonisin B1 hepatotoxicity in mice via modulation of sphingosine kinase.

Fumonisin B(1), a mycotoxin, is an inhibitor of ceramide synthase causing marked dysregulation of sphingolipid metabolism in cells. This mycotoxin causes accumulation of free sphingoid bases (sphingosine and dihydrosphingosine or sphinganine) and their metabolites, important messengers involved in signal transduction leading to either cell survival or death. Free sphingoid bases are known apoptotic molecules whereas sphingosine 1-phosphate is protective. We previously reported that fumonisin B(1) caused sphingosine kinase (SPHK) induction along with the increase of serine palmitoyltransferase (SPT). Fumonisin B(1) also increased inducible nitric oxide synthase (iNOS) expression. In the current study we employed a mouse strain with the targeted deletion of iNOS gene (Nos-KO) to evaluate the role of nitric oxide (NO) on fumonisin B(1)-induced hepatotoxicity. The Nos-KO mice exhibited increased hepatotoxicity after subacute fumonisin B(1) exposure compared to their wild type counterparts, the liver regeneration was lower in Nos-KO compared to that in the WT mice. Increased hepatotoxicity in Nos-KO was not related to the extent of free sphingoid base accumulation after fumonisin B(1) treatment; however, it was accompanied by a lack of fumonisin B(1)-induced SPHK induction. The fumonisin B(1)-induced SPT was unaffected by lack of iNOS gene. Deletion of iNOS gene did not prevent fumonisin B(1)-dependent induction of inflammatory cytokines, namely tumor necrosis factor alpha, interferon gamma and interleukin-12. The lack of fumonisin B(1)-induced SPHK induction in Nos-KO was supported by a similar effect on phosphorylated metabolites of sphingoid bases; the equilibrium between sphingoid bases and their phosphates is maintained by SPHK. We therefore conclude that iNOS induction produced by fumonisin B(1) modulates SPHK activity; the lack of iNOS prevents generation of sphingosine 1-phosphate and deprives cells from its protective effects.

Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides-induced seedling disease.

The fungus Fusarium verticillioides infects maize and produces fumonisins, inhibitors of ceramide synthase. Seeds of the cultivar Silver Queen were inoculated with fumonisin-producing or non-fumonisin-producing strains of F. verticillioides. Leaf lesion incidence and severity of effects on root and stalk growth were significantly correlated with fumonisin in roots and disruption of sphingolipid metabolism in roots. Uninoculated seeds grown in soil watered with solutions of fumonisin B1 exhibited above-ground symptoms indicative of F. verticillioides-induced seedling disease and dose-dependent reduction in root mass that was inversely correlated with fumonisin B1, sphingoid bases, and sphingoid base 1-phosphates in roots. There was also evidence of an adaptive response to disrupted sphingolipid metabolism in both the virulence and watering assays, suggesting induction of pathways responsible for metabolism of sphingoid base 1-phosphates after prolonged exposure. The results suggest that fumonisin, and its effects on sphingolipids, could contribute to all aspects of F. verticillioides maize seedling disease.

Chronic aflatoxin exposure in children living in Bhaktapur, Nepal: Extension of the MAL-ED study.

Exposure to aflatoxin, a mycotoxin common in maize and groundnuts, has been associated with childhood stunting in sub-Saharan Africa. In an effort to further our understanding of growth impairment in relation to mycotoxins and other risk factors, biospecimens from a cohort of children enrolled in the Bhaktapur, Nepal MAL-ED study were assessed for aflatoxin exposure at 15, 24, and 36 months of age. Exposure was assessed through a well-established serum biomarker, the AFB1-lysine adduct. In this manuscript, the levels of aflatoxin exposure in the Nepal cohort were compared with those observed in aflatoxin studies, with child growth parameters as a health outcome. Results from this preliminary analysis demonstrated chronic aflatoxin exposure in children residing in Bhaktapur with a geometric mean of 3.62 pg AFB1-lysine/mg albumin. The range of exposure in this population is similar to those in African populations where associations with aflatoxin biomarkers and poor child growth have been observed. Future work will analyze the relationships between aflatoxin levels, growth, and other risk factors collected by the MAL-ED study.

Differential sensitivity of rat kidney and liver to fumonisin toxicity: organ-specific differences in toxin accumulation and sphingoid base metabolism.

Fumonisins (FBs) are mycotoxins in maize and are inhibitors of ceramide synthase (CS), the most likely proximate cause of FB toxicity. In liver and kidney, the primary target organs in FB-fed rats, inhibition of CS results in a marked increase in the ceramide precursor sphinganine (Sa). This study was conducted to investigate the differential time- and dose-dependent changes in Sa, sphingosine (So), sphinganine 1-phosphate (Sa-1-P), and sphingosine 1-phosphate (So-1-P) in kidney, liver, serum, and heart of male Sprague-Dawley rats (3-4 weeks old) fed diets containing 1.1, 13.5, and 88.6 mug/g of total FB for 10 days. The tissues were microscopically examined for the presence and severity of lesions consistent with FB exposure. There was a time- and dose-dependent increase in Sa in both liver and kidney, which was closely correlated with the tissue concentration of fumonisin B(1) (FB(1)) and histopathologic findings. However, the Sa alone greatly underestimated the degree of disruption of sphingolipid metabolism since accumulated Sa and So were quickly metabolized to Sa-1-P and So-1-P as evidenced by large increases in these metabolites in kidney but not in liver. The concentration of FB(1) in liver and kidney that first elicited an increase in Sa was similar in both tissues, however, over time, the kidney accumulated significantly more FB(1) (10x) and total Sa (Sa plus Sa-1-P) compared to liver. Thus, the relative sensitivity of male Sprague-Dawley rat kidney and liver is most likely a consequence of differences in the mechanisms responsible for both FB(1) uptake/clearance and Sa metabolism.

Toxic effects of fumonisin in mouse liver are independent of the peroxisome proliferator-activated receptor alpha.

Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the role of PPARalpha in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPARalpha-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPARalpha-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPARalpha-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPARalpha-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPARalpha-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPARalpha.

Fumonisin production and bioavailability to maize seedlings grown from seeds inoculated with Fusarium verticillioides and grown in natural soils.

The fungus Fusarium verticillioides infects maize and produces fumonisins. The purpose of this study was to determine the ability of F. verticillioides to produce fumonisins in synthetic and natural soils and their biological availability to maize roots. Maize seeds were inoculated with a pathogenic strain of F. verticillioides (MRC826) and planted in synthetic and three different natural soils. There were statistically significant reductions in stalk weight and root mass and increased leaf lesions in the MRC826-treated seedlings in all soil types. Fumonisins were detected in all of the soils of seedlings grown from MRC826-inoculated seeds. The fumonisin produced in the soils was biologically available to seedlings as demonstrated by the statistically significant elevation of free sphingoid bases and sphingoid base 1-phosphates in their roots. These results indicate that F. verticillioides produced fumonisins in the autoclaved synthetic and natural soils and that the fumonisin produced is biologically available on the basis of evidence of inhibition of ceramide synthase.

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.

Elevated Nuclear and Cytoplasmic FTY720-Phosphate in Mouse Embryonic Fibroblasts Suggests the Potential for Multiple Mechanisms in FTY720-Induced Neural Tube Defects.

FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 in vivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.