ABSTRACT
Detoxification of the maize (Zea mays) antimicrobial compound 2-benzoxazolinone by the fungal endophyte Fusarium verticillioides involves two genetic loci, FDB1 and FDB2, and results in the formation of N-(2-hydroxyphenyl)malonamic acid. Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway. Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X. Overall, 2-benzoxazolinone metabolism involves hydrolysis (FDB1) to produce 2-aminophenol, which is then modified (FDB2) by addition of a malonyl group to produce N-(2-hydroxyphenyl)malonamic acid. If the modification is prevented due to genetic mutation (fbd2), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol. This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce N-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications. Detoxification of these antimicrobials by F. verticillioides apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris.
Subject(s)
Anti-Infective Agents/metabolism , Benzoxazoles/metabolism , Fusarium/metabolism , Zea mays/metabolism , Aminophenols/chemistry , Aminophenols/metabolism , Biotransformation , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fusarium/genetics , Gas Chromatography-Mass Spectrometry , Plant Diseases/microbiology , Zea mays/microbiologyABSTRACT
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.
Subject(s)
Carboxylic Acids/toxicity , Enzyme Inhibitors/toxicity , Fumonisins , Mycotoxins/toxicity , Oxidoreductases/antagonists & inhibitors , Sphingosine/metabolism , Acyltransferases/antagonists & inhibitors , Administration, Oral , Animals , Carboxylic Acids/administration & dosage , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Fatty Acids, Monounsaturated/pharmacology , Intestine, Small/drug effects , Intestine, Small/metabolism , Kidney/drug effects , Kidney/metabolism , LLC-PK1 Cells/drug effects , LLC-PK1 Cells/metabolism , Liver/drug effects , Liver/metabolism , Male , Mice , Mycotoxins/administration & dosage , Serine C-Palmitoyltransferase , Sphingosine/analogs & derivatives , SwineABSTRACT
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.
Subject(s)
Carboxylic Acids/chemistry , Carboxylic Acids/toxicity , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/toxicity , Fumonisins , Liver/enzymology , Mycotoxins/chemistry , Oxidoreductases/antagonists & inhibitors , Acetylation , Animals , Chromatography, High Pressure Liquid , Food Contamination , In Vitro Techniques , Liver/drug effects , Mass Spectrometry , Mycotoxins/toxicity , Rats , Zea mays/microbiologyABSTRACT
The fate of fumonisin B(1) (FB(1)), a mycotoxin found in corn, during the commercial manufacture of fried tortilla chips was studied. FB(1) and hydrolyzed FB(1) (HFB(1)) concentrations in four lots of corn and in the masa, other intermediates, liquid and waste byproducts, and fried chips were determined by HPLC. FB(1) concentrations in the masa and chips were reduced significantly, up to 80% in the fried chips, compared to that in the raw corn. HFB(1) was also found in the masa and chips, but at low concentrations compared to FB(1). LC-MS analyses corroborated HPLC findings and further showed the presence of partially hydrolyzed FB(1) (PHFB(1)), which, like HFB(1), was formed during the nixtamalization (cooking/steeping the corn in alkaline water to make masa) step and found predominantly in the cooking/steeping liquid and solid waste. No significant amounts of N-(carboxymethyl)-FB(1) or N-(1-deoxy-D-fructos-1-yl)-FB(1), indicative of fumonisin-sugar adduct formation, were found. Thus, FB(1) is removed from corn and diverted into liquid and waste byproducts during the commercial production of fried tortilla chips. Nixtamalization and rinsing are the critical steps, whereas grinding, sheeting, baking, and frying the masa had little effect.
Subject(s)
Carboxylic Acids/analysis , Fumonisins , Zea mays/microbiology , Carcinogens, Environmental , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Cooking , Food Analysis , Food Contamination , HydrolysisABSTRACT
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.
Subject(s)
Carboxylic Acids/toxicity , Fumonisins , Kidney/drug effects , Liver/drug effects , Mycotoxins/toxicity , Rodent Diseases/etiology , Animals , Apoptosis/drug effects , Biomarkers , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacokinetics , Food Contamination , Fusarium/chemistry , Fusarium/pathogenicity , Humans , Mycotoxins/chemistry , Mycotoxins/pharmacokinetics , No-Observed-Adverse-Effect Level , Organ Size/drug effects , Rats , Reproduction/drug effects , Rodent Diseases/pathology , Sphingolipids/metabolism , Zea mays/microbiologyABSTRACT
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.
Subject(s)
Carboxylic Acids/toxicity , Carcinogens, Environmental/toxicity , Ceramides/biosynthesis , Fumonisins , Mycotoxins/toxicity , Sphingolipids/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Animals , Apoptosis/drug effects , Enzyme Inhibitors/metabolism , Fusarium , Lipid Peroxidation/drug effects , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/physiopathology , Oxidoreductases/metabolism , Signal TransductionABSTRACT
Of about 10 billion bushels of corn that are grown each year in the United States, less than 2% is processed directly into food products, and about 18% is processed into intermediates such as high-fructose corn syrup, ethanol, and cornstarch. The vast majority of the annual crop is used domestically for animal feed (60%), and about 16% is exported. Thus, any program for controlling residues of fumonisin (FB) in food must recognize that most of the crop is grown for something other than food. Studies on the effects of wet milling on FB residues found these residues nondetectable in cornstarch, the starting material for high-fructose corn syrup and most other wet-milled food ingredients. Similar effects are noted for the dry-milling process. FB residues were nondetectable or quite low in dry flaking grits and corn flour, higher in corn germ, and highest in corn bran. Extrusion of dry-milled products reduces FB concentrations by 30-90% for mixing-type extruders and 20-50% for nonmixing extruders. Cooking and canning generally have little effect on FB content. In the masa process measurable FB is reduced following the cooking, soaking, and washing steps, with little conversion of FB to the hydrolyzed form. Sheeting, baking, and frying at commercial times and temperatures generally have no effect. In summary, all available studies on the effects of processing corn into food and food ingredients consistently demonstrate substantial reductions in measurable FB. No studies have shown a concentration in FB residues in food products or ingredients.
Subject(s)
Carboxylic Acids/analysis , Carcinogens, Environmental/analysis , Food Handling/methods , Food Microbiology , Fumonisins , Mycotoxins/analysis , Zea mays/microbiology , Cooking , Dietary Fiber , Flour , Seeds/microbiologyABSTRACT
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.
Subject(s)
Aflatoxin B1/toxicity , Carboxylic Acids/toxicity , Carcinogens/toxicity , Fumonisins , Liver Neoplasms, Experimental/chemically induced , Methylnitronitrosoguanidine/toxicity , Mycotoxins/toxicity , Sphingosine/analogs & derivatives , Air Sacs/drug effects , Air Sacs/pathology , Animals , Carcinogenicity Tests , Diet , Drug Synergism , Kidney Neoplasms/chemically induced , Kidney Neoplasms/pathology , Liver/drug effects , Liver/metabolism , Liver Neoplasms, Experimental/pathology , Oncorhynchus mykiss , Sphingosine/metabolism , Stomach Neoplasms/chemically induced , Stomach Neoplasms/pathologyABSTRACT
Fumonisin B1 (FB1), a potent mycotoxin prevalent in corn and cereals, causes a variety of toxic effects in different mammalian species. The biochemical responses of FB1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a possible involvement of tumor necrosis factor alpha (TNFalpha). To further characterize the role of TNFalpha, toxic response to FB1 was investigated in male C57BL/6J mice (WT) and a corresponding TNFalpha receptor knockout (TRK) strain, genetically modified to lack the TNFalpha1b receptor. The hepatotoxic effects of 5 daily injections of 2.25 mg/kg per day of FB1 were observed in WT but were reduced in TRK, evidenced by circulating alanine aminotransferase and aspartate aminotransferase levels and histopathological evaluation of the tissue. FB1 induced TNFalpha expression in the livers of both WT and TRK mice to a similar extent (3-4 fold over control); however, a corresponding increase of cellular NFkappaB, expected after the downstream cellular signaling of TNFalpha, was noted only in the WT. Accumulation of liver sphingosine after FB1 treatment was similar in both WT and TRK, but the FB1-induced increases in liver sphinganine and kidney sphingosine and sphinganine were lower in TRK than in WT. Results emphasized the role of TNFalpha in FB1-induced hepatotoxicity in mice and the possible relationship of sphingoid base accumulation and TNFalpha induction. Moreover, the presence of TNFalpha receptor 1b appears to be important in mediating the hepatotoxic responses of TNFalpha and FB1 in mice.
Subject(s)
Antigens, CD/genetics , Carboxylic Acids/toxicity , Fumonisins , Mycotoxins/toxicity , Receptors, Tumor Necrosis Factor/genetics , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Body Weight/drug effects , Cytosol/chemistry , Liver/drug effects , Liver/metabolism , Liver Function Tests , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/biosynthesis , NF-kappa B/genetics , Organ Size/drug effects , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type II , Sphingolipids/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Fumonisins and fusaric acid (FA) are mycotoxins produced by Fusarium moniliforme and other Fusarium which grow on corn. Fumonisins cause animal toxicities associated with F. moniliforme and, like F. monliforme, they are suspected human oesophageal carcinogens. Toxic synergism was obtained by simultaneous administration of FA and fumonisin B1 to chicks in ovo. To determine the effect of FA on in vivo toxicity of F. moniliforme culture material (CM), male rats (12 groups, n = 5/ group) were fed diets containing 0.025, 0.10 or 2.5% CM (providing dietary levels of 3.4, 18.4 or 437 ppm fumonisins, respectively) to which, at each CM level, 0, 20, 100 or 400 ppm FA were added. Additionally, an FA control group was fed 400 ppm FA only and an untreated control group was given neither FA nor culture material. Apoptosis and other effects consistent with those caused by fumonisins were present in the kidneys of animals fed 0.025% or more CM and in the livers of animals fed 2.5% CM. FA was without effect. No differences between the untreated and FA control groups were noted and no differences among the four groups (0-400 ppm FA) fed 0.025% CM, the four groups fed 0.10% CM or the four groups fed 2.5% CM were apparent. Thus, FA exerted no synergistic, additive or antagonistic effects on the subchronic in vivo toxicity of fumonisin-producing F. moniliforme.
Subject(s)
Carboxylic Acids/toxicity , Fumonisins , Fusaric Acid/toxicity , Fusarium , Mycotoxins/toxicity , Administration, Oral , Animals , Body Weight , Brain/drug effects , Cholesterol/blood , Diet , Dose-Response Relationship, Drug , Eating , Enzymes/blood , Heart/drug effects , Kidney/drug effects , Kidney/metabolism , Liver/drug effects , Male , Mycotoxins/administration & dosage , Organ Size , Rats , Rats, Sprague-Dawley , Sphingolipids/analysis , Triglycerides/bloodABSTRACT
Fumonisin B1 (FB1) is a common contaminant of corn worldwide and is responsible for several diseases of animals. In the preparation of tortillas, corn is treated with lime (producing nixtamal) that when heated hydrolyzes at least a portion of the FB1 to the aminopentol backbone (AP1), another known toxin. This study analyzed the amounts of FB1 and AP1 in tortillas and nixtamal from two communities in the central highlands of Guatemala where corn is a major dietary staple (Santa Maria de Jesus, Sacatepequez, and Patzicia, Chimaltenango). The amounts of FB1 and AP1 in tortillas from Santa Maria de Jesus were, respectively, 0.85 +/- 2.0 and 26.1 +/- 38.5 microg/g dry weight (mean +/- SD), and from Patzicia were 2.2 +/- 3.6 and 5.7 +/- 9.4 microg/g dry weight. Less than 6% of the tortillas from both locations contained > or = 10 microg FB1/g dry weight; whereas, 66% of the samples from Santa Maria de Jesus and 29% from Patzicia contained > or = 10 microg AP1/g dry weight. The highest amount of AP1 (185 microg/g dry weight) was found in tortillas from Santa Maria de Jesus. The highest amounts of FB1 were 6.5 and 11.6 microg/g dry weight in tortillas from Santa Maria de Jesus and Patzicia, respectively. The mean concentration of FB1 in nixtamal was significantly higher in Santa Maria de Jesus compared to Patzicia. Surprisingly, AP1 was not detected in any of the nixtamal samples. The human impact of exposure to these amounts of fumonisins is not known. However, based on findings with other animals, where corn is a dietary staple, long-term consumption of FB1 and AP1 (especially at > or = 10 microg/g of the diet) may pose a risk to human health.
Subject(s)
Carboxylic Acids/analysis , Carcinogens, Environmental/analysis , Food Contamination , Fumonisins , Zea mays/chemistry , Calcium Compounds , Food Preservation , Guatemala , OxidesABSTRACT
Fumonisin B1 (FB1) is a frequently encountered mycotoxin that inhibits ceramide synthase, the enzyme that acylates sphinganine, sphingosine and other "sphingoid" bases. Exposure of rats, rabbits, pigs and nonhuman primates to fumonisin-contaminated feed elevates sphingoid base amounts in urine; therefore, this study examined the time course and reversibility of these changes. When an AIN-76 diet supplemented with >/=5 microg FB1/g was fed to male Sprague-Dawley rats, there was a significant increase in sphinganine (ca. 50-fold in urine from rats fed 50 microg FB1/g diet) and smaller changes in sphingosine within 5 to 7 d, compared to rats fed the same diet without FB1. No change occurred in sphingoid bases upon feeding 1 microg FB1/g for up to 60 d. When rats were fed FB1 (10 microg FB1/g diet for 10 d), then changed to the same diet minus FB1, urinary sphingoid bases returned to normal within 10 d. However, if the rats were fed 10 microg FB1/g for 10 d, then changed to 1 microg FB1/g, the amounts of sphingoid bases in urine were the same as for rats that were continuously fed 10 microg FB1/g. These results establish that consumption of FB1 causes dose-dependent and reversible elevations in the amounts of urinary sphingoid bases. The finding that 1 microg FB1/g (which does not, alone, alter urinary sphingoid bases) will sustain the elevation caused by previous exposure to 10 microg FB1/g raises the possibility that even low levels of fumonisins could be deleterious when an animal is occasionally exposed to higher amounts.
Subject(s)
Carboxylic Acids/pharmacology , Fumonisins , Sphingosine/analogs & derivatives , Sphingosine/urine , Animals , Carboxylic Acids/administration & dosage , Diet , Dose-Response Relationship, Drug , Kidney/metabolism , Liver/metabolism , Male , Rats , Rats, Sprague-Dawley , Sphingosine/blood , Sphingosine/metabolism , Time FactorsABSTRACT
Fumonisins are mycotoxins produced by several species of Fusaria. They are found on corn and in corn-based products, can cause fatal illnesses in some animals and are suspected human esophageal carcinogens. Fumonisins are believed to cause toxicity by blocking ceramide synthase, a key enzyme in sphingolipid biochemistry which converts sphinganine (or sphingosine) and fatty acyl CoA to ceramide. Relatively few fungal species have been evaluated for their ability to produce fumonisins. Fewer have been studied to determine if they produce ceramide synthase inhibitors, whether fumonisin-like structures or not, therefore potentially having toxicity similar to fumonisins. We analyzed corn cultures of 49 isolates representing 32 diverse species of fungi for their ability to produce fumonisins. We also evaluated the culture extracts for ceramide synthase activity. Only cultures prepared with species reported previously to produce fumonisins--Fusarium moniliforme and F. proliferatum--tested positive for fumonisins. Extracts of these cultures inhibited ceramide synthase, as expected. None of the other fungal isolates we examined produced fumonisins or other compounds capable of inhibiting ceramide synthase. Although the fungi we selected for these studies represent only a few of the thousands of species that exist, they share the commonality that they are frequently associated with cereal grasses, including corn, either as pathogens or as asymptomatic endophytes. Thus, these results should be encouraging to those attempting to find ways to genetically manipulate fumonisin-producing fungi, to make corn more resistant, or to develop biocontrol measures because it appears that only a relatively few fungal contaminants of corn can produce fumonisins.
Subject(s)
Fungi/metabolism , Mycotoxins/biosynthesis , Sphingolipids/biosynthesis , Animals , Biological Assay , Chromatography, High Pressure Liquid , Fungi/growth & development , Fungi/isolation & purification , Humans , Liver/metabolism , Mycotoxins/pharmacology , Oxidoreductases/antagonists & inhibitors , Poaceae/microbiology , Rats , Zea mays/microbiologyABSTRACT
The purpose of this study was to determine the ability of the fungal serine palmitoyltransferase (SPT) inhibitor, myriocin, to prevent the anti-proliferative and cytotoxic effects of fumonisin B(1) in cultured pig kidney epithelial cells, LLC-PK(1). In an earlier study with LLC-PK(1) cells, ß-chloroalanine (a nonspecific SPT inhibitor) was found to inhibit the fumonisin-induced accumulation of free sphinganine by >90% but only partially reversed (50-60%) fumonisin's antiproliferative and cytotoxic effects. ß-Chloroalanine is not the ideal SPT inhibitor for this type of study because it also inhibits other pyridoxal 5'-phosphate-dependent enzymes. A potent and selective fungal SPT inhibitor (myriocin) was partially purified from liquid cultures of Isaria (=Cordyceps) sinclairii by a combination of organic extraction and column chromatography. The various fractions were bioassayed for their ability to inhibit fumonisin-induced sphinganine accumulation in LLC-PK(1) cells. The activity in partially purified material was compared to the activity of highly purified myriocin and the results expressed as myriocin equivalents. The estimated IC(50) and IC(95) for inhibition of fumonisin-induced sphinganine accumulation were approximately 1.8 and 22 nM, respectively. The IC(95) concentration of the fungal SPT inhibitor reversed the antiproliferative effects and prevented fumonisin-induced apoptosis after 48 h exposure to 50 µM fumonisin B(1). The SPT inhibitor was also effective at reducing free sphinganine in vivo. Free sphinganine concentration was reduced 60% in kidney of mice injected i.p. with SPT inhibitor plus fumonisin B(1) when compared to fumonisin B(1) alone. The ability of SPT inhibition to reduce fumonisin B(1)-induced sphinganine accumulation in vivo may be useful in the development of therapeutic agents for treatment of animals suspected to have been exposed to toxic levels of fumonisin in feeds.
ABSTRACT
The fumonisin (FB) mycotoxins induce liver injury in all species but induce fatal pulmonary edema (PE) only in pigs. They inhibit ceramide synthase in the sphingolipid biosynthetic pathway. To study the pathogenesis of PE, we examined the early events in the development of FB-induced PE and hepatotoxicity in pigs. Pigs were fed FB-contaminated culture material at 20 mg fumonsin B1 (FB1)/kg body weight/day. Groups of 4 pigs were to be euthanatized on 0, 1, 2, 3, 4, or 5 days after initial exposure to FB or when PE developed. Pigs developed PE beginning on day 3; none survived beyond day 4. Progressive elevations in hepatic parameters, including serum enzymes, bile acids, total bilirubin, and histologic changes, began on day 2. Early histologic changes in the lung (day 2) consisted of perivascular edema followed by interlobular and peribronchial edema. Ultrastructurally, alveolar endothelial cells contained unique accumulations of membranous material in the cytocavitary network beginning on day 2. Marked elevations in sphinganine, sphingosine, and their ratio began on day 1 for all tissues whether affected morphologically (lung, liver) or not (kidney, pancreas). The membranous material in endothelial cells may be accumulations of sphingoid bases with damage to the cytocavitary network. Thus, FB induces early elevations in sphingolipids and hepatic injury, followed by alveolar endothelial damage, which may be the critical event in the pathogenesis of PE in pigs.
Subject(s)
Carboxylic Acids/toxicity , Fumonisins , Liver Diseases/veterinary , Liver/drug effects , Mycotoxins/toxicity , Pulmonary Alveoli/drug effects , Pulmonary Edema/veterinary , Swine Diseases/chemically induced , Animals , Blood Chemical Analysis , Chemical and Drug Induced Liver Injury , Endothelium, Vascular/drug effects , Endothelium, Vascular/ultrastructure , Immunoenzyme Techniques/methods , Liver/metabolism , Liver/pathology , Liver Diseases/metabolism , Liver Diseases/pathology , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/ultrastructure , Male , Pulmonary Alveoli/blood supply , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Pulmonary Edema/chemically induced , Pulmonary Edema/metabolism , Pulmonary Edema/pathology , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Swine , Swine Diseases/metabolism , Swine Diseases/pathologyABSTRACT
Fumonisins are mycotoxins produced by Fusarium moniliforme, F. proliferatum, and related Fusarium species found on corn. They occur naturally in corn-based feeds and foods and are suspected human esophageal carcinogens. Fumonisin B1 (FB1), the most common homologue, causes the animal diseases associated with F. moniliforme. Hepato- and nephrotoxicities, disrupted sphingolipid metabolism, and liver cancer have been found in rats fed FB1. To determine the in vivo effects of diets containing fumonisins B2 (FB2) or B3 or (FB3), male rats were fed culture materials (CM) of FB1 non-producing F. moniliforme isolates to provide low (4.6-6.7 ppm), mid (32-49 ppm) or high (219-295 ppm) dietary levels of either FB2 (FB2CM) or FB3 (FB3CM). Other groups were fed culture material of an FB1 producing isolate (FB1CM) providing 6.9, 53 or 303 ppm total fumonisins (FB1: FB2: FB3 = 1.0: 0.38: 0.15) and a tenth group was fed a control diet having no detectable fumonisins. One-half (n = 5/group) the animals were killed after three weeks, at which time the toxicological and histopathological effects of the three culture materials were similar, mimicked the effects of FB1, and included decreased body weight gains, serum chemical indicators of hepatotoxicity, decreased kidney weights, and apoptosis of hepatocytes and kidney tubular epithelium. FB1CM, FB2CM, and FB3CM affected sphingolipids, causing increased sphinganine to sphingosine ratios (Sa/So) in both liver and kidneys. The remaining animals (n = 5/group0 were fed a control diet for three additional weeks. All body weight and tissue specific effects, including increased Sa/So, induced by the FB2Cm, FB3CM and low level FB1CM diets were absent following the recovery period. Except for mild biliary lesions found in the high dose of FB1CM group and a few apoptotic hepatocytes present in one mid- and two high-dose FB1CM rats, no evidence of toxicity remained in these groups, following the recovery period.
Subject(s)
Carboxylic Acids/metabolism , Carboxylic Acids/toxicity , Fumonisins , Fusarium/metabolism , Fusarium/pathogenicity , Mycotoxins/biosynthesis , Mycotoxins/toxicity , Animals , Humans , Kidney/drug effects , Kidney/pathology , Kidney/physiopathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Organ Size/drug effects , Rats , Rats, Sprague-Dawley , Sphingolipids/metabolism , Weight Gain/drug effects , Zea mays/adverse effects , Zea mays/microbiologyABSTRACT
The purpose of this study was (i) to determine whether pure fumonisin B1 could be incorporated into, recovered, and detected by high-pressure liquid chromatographic analysis from the semipurified Oregon test diet (OTD) used in rainbow trout feeding studies, and (ii) to determine if the incorporated fumonisin B1 was biologically available using the change in free sphingoid bases in liver, kidney, and serum as a mechanism-based biomarker. The results indicate that fumonisin is not easily quantified in the OTD. Recoveries ranged from 12 to 81% of the calculated concentrations based on the fumonisin B1 added to the OTD. However, the fumonisin B1 in the OTD was readily absorbed and biologically active as evidenced by marked increases in free sphinganine in liver, kidney, and serum. The magnitude of the increase in free sphinganine at 100 ppm in the OTD was comparable to that known to be associated with liver toxicity in rats, pigs, and ponies.
Subject(s)
Carboxylic Acids/isolation & purification , Food Microbiology , Fumonisins , Mycotoxins/isolation & purification , Animals , Biological Availability , Carboxylic Acids/analysis , Carboxylic Acids/pharmacokinetics , Chromatography, High Pressure Liquid , Food, Formulated , Oncorhynchus mykiss , Rats , Sphingosine/analogs & derivatives , Sphingosine/analysisABSTRACT
Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro- and threo-2-amino, 3-hydroxy- (and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with Vmax/Km of 40-125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4-6 for the threo-isomers. Ceramide synthase also acylates AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a Vmax/Km of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro- and threo-isomers are acylated with the highest apparent Vmax/Km for the erythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins.