Location: Toxicology & Mycotoxin Research2013 Annual Report
1a. Objectives (from AD-416):
1. Determine the biochemical and molecular basis for the species specificity of fumonisins using animal models. 2. Determine the dietary “no observed effect” and “lowest observed effect levels” for neural tube defect induction and determine the dose-response thresholds for elevation in sphingolipid biomarkers in blood spots and fumonisins in urine using animal models. 3. Determine the relationship between fumonisin consumption, urinary fumonisin (exposure biomarker) and changes in sphingolipids in blood spots (effect biomarker) in human populations consuming corn. 4. Determine the specific mechanism(s) by which fumonisins are readily taken up by corn plant roots and yet have limited translocation into above ground vegetative tissues. 5. Determine the effectiveness of alkaline cooking for reducing the toxic potential of fumonisin-contaminated whole kernel corn.
1b. Approach (from AD-416):
1. Conduct dose-response studies to determine the minimum oral dose of FB1 that disrupts sphingolipid metabolism and induces toxicity (increased apoptosis) in rat kidney and mouse liver. 2. Conduct dose-response studies in susceptible mouse strains to determine the thresholds for changes in biomarkers of exposure and effect and induction of neural tube defects. 3. Conduct epidemiological studies to identify humans consuming large amounts of corn-based foods in communities where FB is infrequently detected and frequently detected and sample and analyze urine (FBs) and blood spots (sphingolipids). 4. Conduct dose response studies to determine FB1 affects on plant transpiration and levels of sphingoid bases and their 1-phosphates in roots and aerial tissues in FB1-sensitive and -insensitive genotypes of corn. 5. Utilize FB-contaminated whole kernel corn to determine the processing conditions that maximize FB1 reduction using chemical analysis and in vivo animal bioassays.
3. Progress Report:
Objective 1: When fumonisin (FB) inhibits ceramide biosynthesis in rats and mice, the biochemical responses in target organs are different. In rat, the lipid sphinganine accumulates and is metabolized to its 1-phosphate, which accumulates in kidney to high levels. Accumulation of this metabolite is the cause of the renal toxicity in rats. In mouse liver/kidney there is accumulation of 1-deoxysphinganine. We believe 1-deoxysphinganine is an important biochemical effector of liver toxicity in mice. These results provide evidence for the species and target organ specificity and will provide insight into the human targets. Objective 2: We are conducting studies on the role of dietary folate in induction of neural tube defects (NTD) in mouse. Studies confirm NTD induction by fumonisin B1 (FB1) is reduced in NTD-sensitive LM/Bc mice when fed folate deficient diets for five weeks prior to mating. The protection is associated with an 80 percent reduction of maternal red blood cell folate in mice fed the deficient diet. Based on our earlier work on interactions between folate and FB1, the finding is counterintuitive and suggests the activity of folate in this mouse model with FB1 treatment is much more complicated than previously believed. Objective 3: A survey of fumonisin (FB) contamination in corn in Guatemala was completed. Corn samples (640) were analyzed for FB and aflatoxins (AFB). High levels of AFB and FB were detected in corn from the Petén. High levels of FB, but not AFB, were detected in the Chiquimula and Santa Rosa and very low levels of FB and AFB were detected in the Sacatepéquez. Blood (n=390) and urine (n=390), as well as corn (n=30) for human consumption, were analyzed from Sacatepéquez, Chiquimula, and Santa Rosa. Analysis of the urinary FB biomarker confirmed low exposure in Sacatepéquez and high exposure in Chiquimula and Santa Rosa. The sphingolipid biomarkers in blood are being analyzed. Objective 4: An experimental system was developed to assess the physiological responses and molecular/cellular mechanisms that confer sensitivity/insensitivity to fumonisin B1 (FB1) in maize. Utilizing mutant strains of Fusarium verticillioides, we demonstrated that FB1 accumulates in leaves of the susceptible cultivar Silver Queen without colonization of aerial tissues. Wild-type F. verticillioides colonized leaves and accumulated FB1. Thus, in plant-fungal interactions with the susceptible cultivar, a mechanism for FB1 accumulation exists apart from fungal colonization of leaf. Insensitive inbreds, B73 and W23, accumulate very little FB1 in the leaves when inoculated with wild type, suggesting these maize genotypes are more insensitive, in part, due to less accumulation of FB1. They may suppress FB1 production by the fungus. Further study comparing maize genotypes will identify mechanistic differences. Objective 5: Studies to optimize the nixtamalization process, that will determine the minimum processing procedures (i.e., number and volume of rinses) needed to achieve a significant protective effect and define fumonisin concentrations or other factors limiting reductions, are pending the identification of a suitable source of corn.
1. Survey of Guatemalan maize reveals high levels of aflatoxins and fumonisins in some departamentos (equivalent to counties in the U.S.A.). A total of 640 corn samples from all 22 departamentos of Guatemala were analyzed for both fumonisins and aflatoxins. Very high levels of aflatoxins and fumonisins were detected in corn from the Departamento of Petén. High levels of fumonisins, but not aflatoxins, were also detected in the Departamentos of Chiquimula and Santa Rosa and very low levels of fumonisins and aflatoxins were detected in the highland Departamento of Sacatepéquez. Human urine (n=390), as well as corn (n=30) for human consumption, were collected and analyzed from Sacatepéquez (low fumonisin exposure), Chiquimula (high fumonisin exposure), and Santa Rosa (high fumonisin exposure). Analysis of the urinary fumonisin exposure biomarker confirmed low exposure in Sacatepéquez and high exposure in Chiquimula and Santa Rosa. The results of this study will allow us to validate the usefulness of mechanism-based biomarkers. This is important because the ability to predict the level of fumonisin intake that disrupts sphingolipid metabolism in humans is a necessary first step towards identifying any role for fumonisin exposure in human disease. This study was conducted in collaboration with the Centro de Investigaciones en Nutrición y Salud in Guatemala (CIENSA), Creighton University, and Duke University.
2. Dietary folate modulates neural tube defect induction in LM/Bc mouse model. Dietary folate modulates the induction of neural tube defects by fumonisin B1 in the sensitive LM/Bc mouse model. Consumption of folate deficient diet, beginning five weeks before mating, reduced maternal red blood cell folate concentrations by about 80 percent, compared to mice consuming folate replete diet, and did not significantly reduce the number of phenotypically normal fetuses in each litter. It also protected against neural tube defect induction by fumonisin B1, as fewer litters of folate deficient females had neural tube defects and, within these litters, fewer fetuses had neural tube defects. Modulation of neural tube defect induction by fumonisin B1 likely involves interactions between sphingolipid and folate metabolic pathways.
3. Spores are necessary for systemic colonization of corn, yet fumonisins can accumulate independently of the fungus. The mycotoxigenic fungus Fusarium verticillioides produces fumonisins that are accumulated in the vegetative tissues of the corn plant, as well as in the developing kernels. To demonstrate systemic movement of the fumonisins in seedlings, we showed that infection by the fungus is necessary for accumulation of the fumonisins in the leaves. Interestingly, fumonisin B1 is preferentially accumulated compared to fumonisin B2 or B3. Furthermore, exposing seedling roots to fumonisin B1 alone does not result in movement of the toxin to the leaves since infection is required. Yet, the fungus does not need to be locally present in order for fumonisin to accumulate. By utilizing mutants incapable of producing spores, we showed that spores are necessary for systemic colonization, since mutants inoculated on seed were not able to colonize the stem or leaves of the corn plant, but the non-infected leaves still accumulated fumonisins. These data will allow us to better understand the dynamics of mycotoxin movement and accumulation during fungal infection of the corn plant.
Gelineau-Van Waes, J., Rainey, M.A., Maddox, J.R., Voss, K.A., Sachs, A.J., Gardner, N.M., Wilberding, J.D., Riley, R.T. 2012. Increased sphingoid base-1-phosphates and failure of neural tube closure after exposure to fumonisin or FTY720. Birth Defects Research Part A: Clinical and Molecular Teratology. 94(10):790-803.