Fusarium verticillioides: Advancements in Understanding the Toxicity, Virulence, and Niche Adaptations of a Model Mycotoxigenic Pathogen of Maize.

The importance of understanding the biology of the mycotoxigenic fungus Fusarium verticillioides and its various microbial and plant host interactions is critical given its threat to maize, one of the world's most valuable food crops. Disease outbreaks and mycotoxin contamination of grain threaten economic returns and have grave implications for human and animal health and food security. Furthermore, F. verticillioides is a member of a genus of significant phytopathogens and, thus, data regarding its host association, biosynthesis of secondary metabolites, and other metabolic (degradative) capabilities are consequential to both basic and applied research efforts across multiple pathosystems. Notorious among its secondary metabolites are the fumonisin mycotoxins, which cause severe animal diseases and are implicated in human disease. Additionally, studies of these mycotoxins have led to new understandings of F. verticillioides plant pathogenicity and provide tools for research into cellular processes and host-pathogen interaction strategies. This review presents current knowledge regarding several significant lines of F. verticillioides research, including facets of toxin production, virulence, and novel fitness strategies exhibited by this fungus across rhizosphere and plant environments.

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.

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.

Evidence for fumonisin inhibition of ceramide synthase in humans consuming maize-based foods and living in high exposure communities in Guatemala.

SCOPE: Fumonisin (FB) occurs in maize and is an inhibitor of ceramide synthase (CerS). We determined the urinary FB1 (UFB1 ) and sphingoid base 1-phosphate levels in blood from women consuming maize in high and low FB exposure communities in Guatemala. METHODS AND RESULTS: FB1 intake was estimated using the UFB1 . Sphinganine 1-phosphate (Sa 1-P), sphingosine 1-phosphate (So 1-P), and the Sa 1-P/So 1-P ratio were determined in blood spots collected on absorbent paper at the same time as urine collection. In the first study, blood spots and urine were collected every 3 months (March 2011 to February 2012) from women living in low (Chimaltenango and Escuintla) and high (Jutiapa) FB exposure communities (1240 total recruits). The UFB1 , Sa 1-P/So 1-P ratio, and Sa 1-P/mL in blood spots were significantly higher in the high FB1 intake community compared to the low FB1 intake communities. The results were confirmed in a follow-up study (February 2013) involving 299 women living in low (Sacatepéquez) and high (Santa Rosa and Chiquimula) FB exposure communities. CONCLUSIONS: High levels of FB1 intake are correlated with changes in Sa 1-P and the Sa 1-P/So 1-P ratio in human blood in a manner consistent with FB1 inhibition of CerS.

A blood spot method for detecting fumonisin-induced changes in putative sphingolipid biomarkers in LM/Bc mice and humans.

Fumonisins (FB) are mycotoxins found in maize. They are hypothesised risk factors for neural tube defects (NTDs) in humans living where maize is a dietary staple. In LM/Bc mice, FB1-treatment of pregnant dams induces NTDs and results in increased levels of sphingoid base 1-phosphates in blood and tissues. The increased level of sphingoid base 1-phosphates in blood is a putative biomarker for FB1 inhibition of ceramide synthase in humans. Collection of blood spots on paper from finger sticks is a relatively non-invasive way to obtain blood for biomarker analysis. The objective of this study was to develop and validate in an animal model, and ultimately in humans, a method to estimate the volume of blood collected as blood spots on absorbent paper so as to allow quantification of the molar concentration of sphingoid base 1-phosphates in blood. To accomplish this objective, blood was collected from unexposed male LM/Bc and FB1-exposed pregnant LM/Bc mice and humans and applied to two types of absorbent paper. The sphingoid base 1-phosphates, absorbance at 270 nm (A270), and total protein content (Bradford) were determined in the acetonitrile:water 5% formic acid extracts from the dried blood spots. The results show that in both mouse and human the A270, total protein, and blood volume were closely correlated and the volume of blood spotted was accurately estimated using only the A270 of the extracts. In mouse blood spots, as in tissues and embryos, the FB1-induced changes in sphingolipids were correlated with urinary FB1. The half-life of FB1 in the urine was short (<24 h) and the elevation in sphingoid base 1-phosphates in blood was also short, although more persistent than the urinary FB1.

In vivo formation of N-acyl-fumonisin B1.

Fumonisins are fungal toxins found in corn and in corn-based foods. Fumonisin B1 (FB1) is the most common and is toxic to animals, causes cancer in rodents, and is a suspected risk factor for cancer and birth defects in humans. The hydrolyzed form of FB1 (HFB1) also occurs in foods and is metabolized by rats to compounds collectively known as N-acyl-HFB1 (also known as N-acyl-AP1). N-acyl-HFB1 is structurally similar to ceramides, metabolites which have important structural and signaling functions in cells. FB1 is N-acylated in vitro to ceramide-like metabolites which, like FB1, are cytotoxic. However, metabolism of FB1 and inhibition of ceramide synthase by its metabolites in vivo has not been demonstrated. Male rats were dosed ip with 0.5, 1, or 2 mg/kg body weight FB1 on five consecutive days and the liver and kidney thereafter processed for chemical analysis. N-acyl derivatives of fumonisin B1 were identified for the first time in these principal target organs of FB1 in rats, at levels up to 0.4 nmol/g tissue using mass spectrometry. The N-acyl chain length of the metabolites varied in a tissue-dependent manner with C16 derivatives predominating in the kidney and C24 derivatives being prevalent in the liver. The toxicological significance of N-acyl-fumonisins is not known and warrants investigation.

Fumonisin B1 induced neural tube defects were not increased in LM/Bc mice fed folate-deficient diet.

SCOPE: Fumonisin B1 (FB1 ) is found in corn-based foods and is a possible risk factor for neural tube defects (NTDs). The mechanism(s) underlying NTD induction by FB1 in LM/Bc mice is not understood; however, evidence suggests disrupted folate transport is involved. METHODS AND RESULTS: Female LM/Bc mice were fed folate-sufficient (control) or folate-deficient diet beginning 5 wk before mating, treated with 0 (vehicle), 2.5 or 10 mg/kg FB1 by intraperitoneal injection on embryonic days 7 (E7) and E8, and their fetuses examined on E16. Dose-dependent NTD induction was found in groups fed the control diet: 3 of 13 low-dose and 10 of 11 high-dose litters were affected. Among groups fed folate-deficient diet, NTDs were found only in 4 of 11 high-dose litters. In another trial, consumption of folate-deficient diet also resulted in fewer NTDs at a dose of 10 mg/kg FB1 and reduced maternal red blood cell folate levels by 80%. In utero death did not fully account for the differences in NTD rates. CONCLUSION: Folate deficiency does not exacerbate NTD induction by FB1 in LM/Bc mice. Interactions between folate, other nutritional factors, and FB1 in this mouse model for NTDs are complex and require further investigation.

Urinary fumonisin B1 and estimated fumonisin intake in women from high- and low-exposure communities in Guatemala.

SCOPE: Fumonisin (FB) intake can be high when maize is a dietary staple. We determined (i) urinary FB (UFB) in women consuming maize in high- and low-exposure communities in Guatemala, (ii) the FB levels in maize, (iii) the relationship between UFB and FB intake, and (iv) the relative excretion of UFB1 , UFB2 , and UFB3 . METHODS AND RESULTS: Urine and maize were analyzed for FB for 1 year in three departments. Maize consumption was estimated by an interview questionnaire. Fumonisin B1 , B2 , and B3 (FB1 , FB2 and FB3 ), were detected in 100% of maize samples. FB1 in maize and urine was significantly higher in Jutiapa compared to Chimaltenango or Escuintla. The FB intake paralleled UFB1 in a dose-dependent manner but UFB1 was present in much higher levels than UFB2 or UFB3 compared to maize. CONCLUSION: In Jutiapa, agroecological conditions favored FB production. UFB1 mirrored the estimated FB intake. UFB1 > 0.1 ng/mL resulted in a dose-dependent increase in the risk of exceeding FB intake of 2 µg/kg b.w./day compared to women with no detectable UFB1 . More than 50% exceeded 2 µg/kg b.w./day when UFB1 was >0.5 ng/mL. UFB2 and UFB3 were rarely detected confirming that FB1 is either absorbed better or preferentially excreted in urine.

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.

The kinetics of urinary fumonisin B1 excretion in humans consuming maize-based diets.

SCOPE: Fumonisins (FB) are mycotoxins found in maize. The purpose of this study was to (i) determine the relationship between FB(1) , FB(2) , and FB(3) intake and urinary excretion in humans, (ii) validate a method to isolate urinary FB on C(18) -SPE cartridges for international shipment, and (iii) test the method using samples from Guatemala. METHODS AND RESULTS: Volunteers (n = 10) consumed 206 grams/day of tortillas and biscuits prepared from masa flour and a product containing maize flour. Volunteers estimated their daily urine output and samples were analyzed for FB(1) , FB(2) , and FB(3) and hydrolyzed FB(1) . Only FB(1) was detected in urine suggesting lower absorption of FB(2) and FB(3) . Excretion was highly variable peaking soon after consumption began and decreasing rapidly after consumption stopped. Within 5 days after consumption ended, FB(1) was not detected in urine. In a study with eight volunteers, the average total urinary FB(1) was 0.5% of the intake. FB(1) was detected in 61% (107/177) of the samples collected in Guatemala. CONCLUSION: The results support the use of urinary FB(1) to assess ongoing exposure in population-based studies. However, relating the FB(1) concentration in urine to dietary intake of FB by individual subjects will be complicated due to interindividual variability and the rapidity of clearance.

Extrusion cooking with glucose supplementation of fumonisin-contaminated corn grits protects against nephrotoxicity and disrupted sphingolipid metabolism in rats.

SCOPE: Fumonisin B1 (FB1) is a mycotoxin found in maize and maize-based foods. It causes animal diseases and is a suspected risk factor for cancer and birth defects in humans. Extrusion cooking reduces FB1 concentrations in maize however toxicity caused by unknown degradation or FB1-matrix reaction products might persist. METHODS AND RESULTS: To test the efficacy of extrusion to reduce FB1 toxicity, Fusarium verticillioides fermented corn (= maize) grits (Batch-1= 9.7 ppm FB1; Batch-2= 50 ppm FB1) were extruded without (Batch-1E; Batch-2E) or with 10% glucose supplementation (Batch-1EG; Batch-2EG). FB1 concentrations were reduced 64% (Batch-2E) to 94% (Batch-1EG) after cooking. When the uncooked and processed grits were fed (50% w/w in rodent chow) to rats for up to 8 weeks, FB1 intakes averaged 354, 103, and 25.1 çg/kg body weight/day for Batch-1, Batch-1E and Batch-1EG and 1804, 698, and 222 çg/kg body weight/day for the Batch-2, Batch-2E and Batch-2EG, respectively. Nephrotoxicity including apoptotic lesions and elevated sphingoid base concentrations decreased in a dose-dependent manner in groups fed Batch-1, Batch-1E, Batch-2, Batch-2E, or Batch-2EG and was absent in the Batch-1EG group. CONCLUSION: Extrusion cooking, especially with glucose supplementation, is potentially useful to reduce FB1 concentrations and toxicity of FB1-contaminated maize.

Reduction of fumonisin B1 in corn grits by twin-screw extrusion.

UNLABELLED: This study was designed to investigate the fate of fumonisins in flaking corn grits during twin-screw extrusion by measuring fumonisin B1 (FB1) and its analogs with a mass balance approach. Food grade corn grits and 2 batches of grits contaminated with FB1 at 10 and 50 µg/g by Fusarium verticillioides M-2552 were processed with or without glucose supplementation (10%, w/w) with a twin-screw extruder. Extrusion reduced FB1 in contaminated grits by 64% to 72% without glucose and 89% to 94% with added glucose. In addition, extrusion alone resulted in 26% to 73% reduction in the levels of fumonisin B2 and fumonisin B3, while levels of both mycotoxins were reduced by >89% in extruded corn grits containing 10% glucose. Mass balance analysis showed that 38% to 46% of the FB1 species detected in corn extruded with glucose was N-(deoxy-D-fructos-1-yl)-FB1, while 23% to 37% of FB1 species detected in extruded corn grits with and without added glucose was bound to the matrix. It was also found that the hydrolyzed form of FB1 was a minor species in extruded corn grits with or without added glucose, representing <15% of the total FB1 species present. Less than 46% of FB1 originally present in corn grits could be detected in the fumonisin analogues measured in this study. Research is needed to identify the reaction products resulting from extrusion processing of fumonisin-contaminated corn products. PRACTICAL APPLICATION: Twin-screw extrusion is widely used in food industry for its versatility. This technology may reduce the level of fumonisins in corn particularly with added glucose.

Reproductive and sphingolipid metabolic effects of fumonisin B(1) and its alkaline hydrolysis product in LM/Bc mice: hydrolyzed fumonisin B(1) did not cause neural tube defects.

Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins are converted to their hydrolyzed analogs by alkaline cooking (nixtamalization). Both fumonisins and hydrolyzed fumonisins are found in nixtamalized foods such as tortillas, and consumption of tortillas has been implicated as a risk factor for neural tube defects (NTD). Fumonisin B(1) (FB(1)) induced NTD when given (ip) to pregnant LM/Bc mice; however, neither the NTD induction potential of hydrolyzed fumonisin B(1) (HFB(1)) nor its affect on sphingolipid metabolism in pregnant mice have been reported. The teratogenic potential of FB(1) and HFB(1) was therefore compared using the LM/Bc mouse model. Dams were dosed (ip) with 2.5, 5.0, 10, or 20 mg/kg (< or = 49 micromol/kg) body weight (bw) HFB(1) on embryonic day (E)7-E8. Negative and positive control groups were given vehicle or 10 mg/kg (14 micromol/kg) bw FB(1), respectively. The high dose of HFB(1) disrupted sphingolipid metabolism, albeit slightly, but did not cause maternal liver lesions or NTD (n = 8-10 litters per group). In contrast, 10 mg/kg bw FB(1) markedly disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the dams, increased fetal death rates, and decreased fetal weights. Furthermore, NTD were found in all FB(1)-exposed litters (n = 10), and 66 +/- 24% of the fetuses were affected. The findings indicate that HFB(1) does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupts sphingolipid metabolism at doses up to sevenfold higher (micromole per kilogram body weight basis) than the previously reported lowest observed adverse effect level for FB(1).

Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.

Fumonisin B(1) (FB(1)) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB(1) increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C(18)H(40)NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH(3)CH=NH (+)(2)) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-(13)C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK(1) cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-(13)C]palmitate into 1-[(13)C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[(13)C]deoxySa. 1-DeoxySa was elevated in FB(1)-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK(1) and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB(1), substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation.

Reduced toxicity of fumonisin B1 in corn grits by single-screw extrusion.

Corn grits spiked with 30 microg/g fumonisin B1 and two batches of grits fermented with Fusarium verticillioides (batch 1 contained 33 microg/g, and batch 2 contained 48 microg/g fumonisin B1), which were extruded by a single-screw extruder with and without glucose (10%, dry weight basis) supplementation were fed to rats. Control groups were fed uncontaminated grits. Extrusion with glucose more effectively reduced fumonisin B1 concentrations of the grits (75 to 85%) than did extrusion alone (10 to 28%). With one exception, the fumonisin B1-spiked and fermented extrusion products caused moderately severe kidney lesions and reduced kidney weights, effects typically found in fumonisin-exposed rats. Lesions in rats fed the least contaminated grits (batch 1) after extrusion with 10% glucose were, however, significantly less severe and not accompanied by kidney weight changes. Therefore, extrusion with glucose supplementation is potentially useful for safely reducing the toxicity of fumonisins in corn-based products and studies to determine the optimal conditions for its use are warranted.

Listeria monocytogenes infection in pregnant guinea pigs is associated with maternal liver necrosis, a decrease in maternal serum TNF-alpha concentrations, and an increase in placental apoptosis.

Stillbirths and spontaneous abortions can result when pregnant women are exposed to the food borne pathogen, Listeria monocytogenes. Fetuses and neonates account for one-third of the 2500 cases annually. The objectives were to determine the dose dependent trends of immunological and pathological effects in pregnant guinea pigs after infection with L. monocytogenes. Timed pregnant guinea pigs were treated on gestation day (gd) 35 with doses of 10(4) to 10(8) colony forming units (CFUs) and sacrificed on gd 56. Hepatic lesions were found in dams treated with >or=10(5)CFUs. Apoptosis was detected in significantly more placentas from dams treated with >or=10(6)CFUs compared to controls. Maternal serum TNF-alpha concentrations were significantly decreased in all dose groups compared to controls. In conclusion, increases in premature delivery, maternal hepatic effects and placental apoptosis along with a decrease in TNF-alpha concentrations were associated with L. monocytogenes infection in pregnant guinea pigs.

Hydrolyzed fumonisins HFB1 and HFB2 are acylated in vitro and in vivo by ceramide synthase to form cytotoxic N-acyl-metabolites.

Fumonisins B1 and B2 (FB1 and FB2) are the most abundant members of the fumonisins--mycotoxins that are produced by Fusarium verticillioides and are natural inhibitors of ceramide synthase. Their hydrolyzed forms, HFB1 and HFB2 (also called AP1 and AP2) are found in some foods, and they are not only inhibitors of ceramide synthase but also undergo acylation by this enzyme. This study characterized the conversion of HFB1 and HFB2 by ceramide synthase to their respective N-acylated metabolites using rat liver microsomes and palmitoyl-CoA or nervonoyl-CoA as cosubstrates, and examined animals that had been dosed with hydrolyzed fumonisins to ascertain if acylation occurs in vivo. Using an HPLC-MS/MS method that allowed the sensitive and selective detection of the acylation products, both HFB1 and HFB2 were found to be metabolized in vitro to nervonoyl- or palmitoyl-HFB1 and -HFB2 (i.e. C24:1-HFB1/2 and C16-HFB1/2, respectively). The apparent vmax was considerably higher for formation of C24:1HFB1 (157 pmol/min/mg protein) than for formation of C16HFB1 (8.7 pmol/min/mg protein). The acylation products also inhibited ceramide synthase and significantly reduced the number of viable cells in an in vitro [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay using a human colonic cell line (HT29). Furthermore, HPLC-MS/MS analysis of tissues from rats given intraperitoneal doses of HFB1 confirmed that formation of N-acyl-HFB1 occurs in vivo to produce metabolites with fatty acids of various chain lengths. The contribution of acylated HFB1 and HFB2 metabolites to fumonisin toxicity in vivo warrants further investigation.