Acylation of Naturally Occurring and Synthetic 1-Deoxysphinganines by Ceramide Synthase
FORMATION OF N-PALMITOYL-AMINOPENTOL PRODUCES A TOXIC METABOLITE OF HYDROLYZED FUMONISIN, AP1, AND A NEW CATEGORY OF CERAMIDE SYNTHASE INHIBITOR*

Hans-Ulrich HumpfDagger §, Eva-Maria Schmelz, Filmore I. Meredithparallel , Hubert Vesper, Teresa R. Vales, Elaine Wang, David S. Menaldino, Dennis C. Liotta, and Alfred H. Merrill Jr.§

From the Dagger  Lehrstuhl für Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany,  Departments of Chemistry and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, and parallel  Toxicology & Mycotoxin Research Unit, United States Department of Agriculture, Agricultural Research Service, Athens, Georgia 30613

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Fumonisins are mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi that are common contaminants of maize and certain other foods (1). Fumonisin B1 (FB1)1 is comprised of a long chain aminopentol (AP1) with two of the side chain hydroxyls esterified to tricarballylic acids (Fig. 1) (2, 3). Although numerous fumonisins have been characterized (1-4), FB1 is usually the most abundant in contaminated food, except when corn has been treated with base to produce masa flour for tortillas, which hydrolyzes FB1 to AP1 (1).


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Fig. 1.   Structures of FB1, the aminopentol AP1, and the corresponding ceramide analogs, N-palmitoyl-FB1 (PFB1) and PAP1.

Fumonisins are responsible for at least two diseases, equine leukoencephalomalacia and porcine pulmonary edema (5, 6). Studies with these and other animals have uncovered a wide spectrum of toxicologic effects, which include hepatotoxicity, nephrotoxicity, neurotoxicity, developmental toxicity, and immunosuppression (and immunostimulation under some conditions) (for reviews, see Refs. 1, 7, and 8). Fumonisins are also hepatocarcinogenic in rats (1) and have been implicated in esophageal cancer in humans in South Africa and China (9-11).

The diverse effects of these mycotoxins appear to be due to inhibition of ceramide synthase, the enzyme that catalyzes the acylation of sphinganine, sphingosine, and other sphingoid bases (12, 13). Inhibition has been shown in vitro with intact cells and for animals exposed to fumonisins (7) and is manifested by up to several hundredfold increases in cellular levels of sphinganine (and sometimes other sphingoid bases) (8, 14, 15). This elevation in sphinganine, a highly bioactive compound, initiates a cascade of cellular alterations that are thought to be largely responsible for the toxicity and carcinogenicity of this mycotoxin (7, 16-19).

Removal of the tricarballylic acids groups from FB1 reduces the potency of inhibition of ceramide synthase by approximately 10-fold (13, 19). This observation, plus the kinetic pattern for inhibition (20), suggests that ceramide synthase recognizes both the AP1 moiety (as a sphingoid base analog) and the tricarballylic acids side chains (presumably, as analogs of the phosphate groups of fatty acyl-CoA) (13). The toxicity of AP1 for cells in culture mirrors this reduced potency (19); however, feeding studies have found that AP1 causes hepatic and renal lesions in rats that are indistinguishable from those caused by FB1 (21, 22), which suggests that the mechanism(s) of action of AP1 versus FB1 are not yet fully understood.

To characterize further the properties of the AP1 backbone, a series of 2-amino-3-hydroxy- (and 3, 5-dihydroxy-) octadecanes have been synthesized, and this report describes the somewhat unexpected finding that these analogs not only inhibit ceramide synthase but also serve as alternative substrates. Because these findings indicated that the aminopentol backbone of fumonisins may be acylated by ceramide synthase, the formation of N-palmitoyl-AP1 (PAP1) was also characterized. Finally, PAP1 was found to be highly toxic for HT29 cells, apparently as a new category of ceramide synthase inhibitor.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The synthetic fumonisin and sphingoid base analogs were prepared by methods that will be described in another publication.2 FB1 was isolated from culture material, and AP1 was synthesized from FB1 by basic hydrolysis, as described by Wilson et al. (23). Radiolabeled compounds were purchased from Amersham Pharmacia Biotech ([1-14C]palmitoyl-coenzyme A) and NEN Life Science Products (D-erythro-[3-3H]sphingosine).

Analytical Methods-- For FAB-MS, a JMS-SX102SX102A/E five sector tandem mass spectrometer (JEOL, Peabody, MA) was used with NBA-Li as matrix. Electrospray analysis was performed on a triple stage quadrupole TSQ 7000 LC-MS/MS system (Finnigan MAT, Bremen, Germany). HPLC/MS separations were carried out on a LiChrospher 60-RP select B column (100 × 2.0 mm inner diameter, 5 µm; Knauer, Berlin, Germany) using a linear water/acetonitrile gradient with 0.05% trifluoracetic acid.

Synthesis of N-Palmitoyl-FB1-- FB1 (1 mg, 1.38 µmol) and 4.4 mg of palmitic anhydride (6.92 µmol) were dissolved in 1 ml of methanol (MeOH):CHCl3 (1:1, v/v) and kept at room temperature for up to 48 h. The reaction was checked by TLC using silica 60 TLC plates (Merck, EM Separations, Gibbstown, NJ) and CHCl3:MeOH:H2O (90:20:0.5) as the developing solvent. To detect fumonisins, the plates were sprayed with a 0.5% p-anisaldehyde solution in MeOH:acetic acid:sulfuric acid (85:10:5), heated to 100 °C for 10 min, and visually examined. The clean-up was carried out by extraction with n-butanol as described for the ceramide synthase assay of FB1 (see below). Results: yield 40-50%; ES-MS: m/z 960.6 [M+H]+.

Synthesis of N-Palmitoyl-AP1 (PAP1)-- AP1 (1.1 mg, 2.7 µmol) and 5.3 mg of palmitic anhydride (10.7 µmol) were dissolved in 1.5 ml of MeOH:CHCl3 (1:1) and kept at room temperature for up to 48 h. The reaction was checked by TLC as described above. The reaction mixture (0.5 ml) was applied to a SiO2 SPE column (500 mg capacity; Waters, Milford, MA), which was preconditioned with CHCl3. The minicolumn was washed with 6 ml of CHCl3 followed by 6 ml of CHCl3/MeOH (95:5). PAP1 was eluted with 6 ml of CHCl3:MeOH (95:10). Results: yield 70-80%; FAB-MS (NBA-Li-matrix): m/z 650.5 [M+Li]+, ES-MS: m/z 644.8 = [M+H]+.

Assays of Ceramide Synthase-- Ceramide synthase was assayed as described by Merrill and Wang (24) by following either acylation of [3H]sphingosine to ceramide (for inhibition studies) or acylation of the sphingoid base analog using a radiolabeled fatty acyl-CoA. For the studies with FB1 or AP1, the reaction mixture (totalling 100 µL) contained 25 mM potassium phosphate buffer, pH 7.4, 0.5 mM dithiothreitol, 50 µM [1-14C]palmitoyl-CoA, varying concentrations of FB1 or AP1 (1-10 µM), and 150 µg of microsomal protein. The enzymatic reaction mixture was incubated in a shaking water bath for 15 min at 37 °C. In the case of AP1 as substrate, the reaction was stopped by the addition of 1 ml of MeOH and 0.5 ml of CHCl3. For lipid extraction, 1 ml of CHCl3 and synthetic PAP1 (~25 µg, as carrier) was added. The reaction mixture was vortexed, approximately 3 ml of slightly basic water was added, and the mixture was vortexed again. The CHCl3 layer was washed, dried, and evaporated as described above. In the case of FB1 as substrate, the reaction was stopped by the addition of 1 ml of CHCl3, then, ~25 µg of synthetic N-palmitoyl-FB1 was added as carrier, followed by 2 ml of slightly basic water. The reaction mixture was vortexed, centrifuged, and the CHCl3 layer was again washed with slightly basic water, dried, evaporated, and analyzed by TLC. The aqueous phase was acidified (to pH 4-5) by adding 0.1 N HCl, and extracted two times with 1 ml of n-butanol. The combined butanol layers were dried over Na2SO4, evaporated to dryness using a Speed Vac (Savant Instruments, Farmingdale, NY) and analyzed by TLC.

Analyses of the Ceramide Synthase Assay Products by TLC-- Samples were dissolved in MeOH:CHCl3 (1:2) and spotted onto type 60 silica TLC plates that were developed using CHCl3:MeOH (90:10, solvent 1) for PAP1, diethylether:MeOH (99:1, solvent 2) for ceramides, CHCl3:MeOH:H2O:acetic acid (90:20:0.5:1, solvent 3) for N-palmitoyl-FB1. After the plates were developed and air dried, the products were visualized by spraying with p-anisaldehyde solution, and the radiolabeled regions of the plate were detected using a Bioscan System 200 Image Scanner (Bioscan Inc., Washington, DC). The regions that migrated coincident with the standards were collected and counted using a detergent-containing scintillation mixture for 10 min in a Beckmann LS 7000 scintillation counter (Beckman Instruments, Palo Alto, CA).

Cell Culture-- HT29 cells (ATCC, Rockville, MD) were seeded at 1 × 105 cells per 100-mm dish (Corning, Cambridge, MA), and grown in 8 ml of Dulbecco's modified Eagle medium (Sigma) supplemented with 10% fetal calf serum (HyClone, Logan, UT), 3.5 g/liter glucose, and 10 ml/liter of a solution of 6.1 mg/ml penicillin G and 10 mg/ml streptomycin (Sigma) in an incubator at 37 °C and a humidified atmosphere of 5% CO2. The medium was changed 24 h after seeding, then the cells were incubated with Dulbecco's modified Eagle's medium containing PAP1 (solubilized in 10 µl of ethanol:dodecane, 98:2; addition of this volume of ethanol:dodecane had negligible effects on cell viability). After 24 h, the cells were harvested with 0.25% trypsin in 0.05% EDTA, and viable cells were determined by trypan blue exclusion.

To determine the effects of fumonisins on cellular sphinganine and ceramides, the cells were treated in the same manner, and the lipids were extracted and analyzed by HPLC as described by Schmelz et al. (19).

Statistical Analyses-- Analyses were conducted in triplicate unless otherwise noted, and the statistical significance of differences between groups was evaluated by the Student's t test using the InStat statistical package (GraphPad Software). The kinetic parameters were derived from linear regression analyses of the data.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Inhibition of Ceramide Synthase by Fumonisin Analogs-- All of the fumonisin analogs inhibited the acylation of D-erythro-[3H]sphingosine (Fig. 2), but were less potent than FB1. These results agree with the previous demonstration that FB1 is more potent than AP1 which, like these analogs, lacks the tricarballylic acid side chains (13). Upon more detailed analyses of the products of the assays, we discovered that the analogs also served as substrates; therefore, the kinetic parameters of acylation of these compounds were analyzed (Fig. 3). Of the six fumonisin analogs tested, XXa had the highest apparent Vmax, which was comparable with the naturally occurring D-erythro-sphinganine and -sphingosine (13, 25). Similarly, the other erythro- analogs (Va and XXb) were acylated reasonably well, and the threo- compounds (Vb, XIVa and XIVb) were also acylated, but with much lower apparent Vmax. Therefore, ceramide synthase does not require a 1-hydroxyl group on the sphingoid base substrate and as has been seen in analyses of the stereoisomers of sphingosine and sphinganine (25) will accommodate both threo- and erythro-stereoisomers, although the latter are better substrates. The ability of this enzyme to acylate these fumonisin analogs raised the possibility that FB1 or AP1 might also be acylated, therefore, this was investigated.


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Fig. 2.   Inhibition of the acylation of [3H]sphingosine by ceramide synthase by fumonisin analogs. Each compound was added at 10 µM to ceramide synthase assays conducted as described under "Experimental Procedures." The percent inhibition is calculated versus the control without inhibitor; the data are shown as mean ± S.E. (n = 3).


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Fig. 3.   Kinetic constants (apparent) for the utilization of sphinganine and various fumonisin analogs by ceramide synthase. Km is expressed in µM AND Vmax in pmol/min/mg of microsomal protein for each of the compounds, R = C11H23.

Preparation of Reference Compounds-- Since palmitoyl-CoA is one of the substrates utilized by ceramide synthase as well as widespread in nature, we used this fatty acid for the chemical and enzymatic syntheses. The N-palmitoyl-FB1 and PAP1 (Fig. 1) were characterized by FAB-MS and electrospray (ES)-MS analysis. The electrospray spectra of N-palmitoyl-FB1 and PAP1 showed strong protonated molecular ions at m/z 960.6 [M+H]+ and m/z 644.8 [M+H]+, respectively, without further fragmentation. Using FAB-MS, only PAP1 gave a strong molecular ion at m/z 650.5 [M+Li]+.

Assays of Ceramide Synthase with AP1 and FB1-- Since PAP1 is similar to natural ceramides in polarity, the products were extracted using CHCl3:MeOH (24); however, N-palmitoyl-FB1 bears two tricarballylic acids groups (with four charged carboxyl groups) and required a different extraction protocol. Briefly, the reaction mixture was extracted with CHCl3 to remove nonpolar lipids, then the aqueous phase was acidified with HCl and extracted with n-butanol to obtain N-palmitoyl-FB1 (for details see "Experimental Procedures"). The success of the extraction was monitored by TLC and MS, and most of the carrier N-palmitoyl-FB1 was found in the n-butanol layer (attempts to purify N-palmitoyl-FB1 by other approaches, such as using reversed phase or anion exchange cartridges, were not successful).

As shown in Fig. 4, incubation of rat liver microsomes with 10 µM AP1 and [1-14C]palmitoyl-CoA produced a radiolabeled product that migrated at the same position on the chromatoplate (Rf ~0.2) as the reference PAP1 (radiolabel was not found in this region of the chromatoplate when AP1 was omitted from the assay). Co-migration of this labeled product and the PAP1 standard on TLC was also obtained using several other solvent systems (not shown). To confirm that this product was PAP1, the assay mixture was extracted and the product analyzed by HPLC-ES-MS/MS using a reversed phase column and a water/acetonitrile gradient. We were able to detect the protonated molecular ion in the single ion monitoring mode at m/z 644.8 [M+H]+. Since only low amounts of PAP1 were produced in these enzymatic syntheses, we were not able to obtain daughter ion spectra; however, the m/z 644.8 [M+H]+ signal showed the same HPLC retention time as the reference compound, which is strong evidence for the identity of this product.


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Fig. 4.   Biosynthesis of PAP1 from AP1 and [1-14C]palmitoyl-CoA by rat liver microsomes. The products of the incubation were resolved on silica gel thin-layer chromatography in the solvent system CHCl3:MeOH (90:10), and the lipids were visualized with p-anisaldehyde (panel A), and the radiolabeled compounds detected by radiometric scanning of the TLC plate (panel B). For further experimental details, see "Experimental Procedures."

The concentration dependence for PAP1 formation fits a simple Michaelis-Menten relationship (Fig. 5). The apparent Km was 3.4 µM and Vmax was 4.0 pmol/min/mg microsomal protein for AP1 (Fig. 5). Thus, the Vmax/Km for AP1 is approximately 1, which is on the same order of magnitude as for the threo-fumonisin analogs (Vb, XIVa and b).


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Fig. 5.   Double reciprocal plot of ceramide synthase activity with varying concentrations of AP1 and 50 µM [1-14C]palmitoyl-CoA. Ceramide synthase activity (in pmol PAP1 formed/min/mg of microsomal protein) was determined as described under "Experimental Procedures."

When 10 µM FB1 was incubated with rat liver microsomes and [1-14C]palmitoyl-CoA, no radiolabel was detected in the region of the N-palmitoyl-FB1 standard, therefore, FB1 was not acylated under these assay conditions.

Effects of PAP1 on HT29 Cells-- To determine whether PAP1 is cytotoxic, this compound was incubated with HT29 cells, a human colonic cell line that is sensitive to FB1 and AP1 (19). All of the tested concentrations of PAP1 (1-50 µM) caused a significant reduction in the number of viable cells within 24 h (cell death was noted as early as 4 h after addition of 50 µM PAP1) (Fig. 6). In other studies (19), we have shown that 50 µM FB1 and AP1 reduced cell number by 50 and 32%, respectively, after 24 h.3 Therefore, PAP1 appears to be considerably more toxic for HT29 cells than either of the parent fumonisin precursors.


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Fig. 6.   Analysis of the toxicity of PAP1 for HT29 cells. The cells were incubated with the shown concentrations of PAP1 for 24 h, then the number of viable cells were counted using a hemocytometer. The control cells received only the solvent carrier (10 µl of ethanol:dodecane, 98:2). The results are shown as mean ± S.E. (n = 3); groups that are significantly different from the control are designated by "*".

Since the toxicity of fumonisins has been associated with the accumulation of sphinganine, the effects of PAP1 on free sphinganine and ceramide were determined (Fig. 7). The elevation in sphinganine induced by 1 µM PAP1 (17-fold versus the no fumonisin control) was the same as that for 50 µM AP1, and the elevation induced by 5 µM PAP1 (148-fold versus the no fumonisin control) was approximately twice the fold increase induced by 50 µM FB1 (68-fold). None of these treatments altered the ceramide levels of the cells over this period (Fig. 7), which is consistent with the sphinganine accumulation arising from inhibition of ceramide synthase, rather than activation of a ceramidase.


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Fig. 7.   Effects of toxic concentrations of FB1, AP1, and PAP1 on the amounts of free sphinganine and ceramide in HT29 cells. The cells were incubated with the shown concentrations of fumonisins as in Fig. 6, and after 24 h the amounts of free sphinganine and ceramide were determined as described under "Experimental Procedures." The results are shown as mean ± S.E. (n = 3); groups that are significantly different from the control are designated by "*". black-square, sphinganine (nmol/106 cells); square , ceramide (nmol/106 cells).

Inhibition of Ceramide Synthase in Vitro by PAP1 and N-Palmitoyl-XIVa-- As predicted by these findings with HT29 cells, PAP1 (and the N-palmitoyl derivative of the synthetic analog that most resembles AP1, XIVa) inhibited ceramide synthase in vitro, as shown in Fig. 8. Both caused ~50% inhibition at ~9 µM; for comparison, FB1 caused 50% inhibition at 6 µM in parallel assays conducted with this enzyme preparation and reagents (data not shown).


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Fig. 8.   Inhibition of ceramide synthase in vitro by N-palmitoyl-XIVa and PAP1. Ceramide synthase was assayed as described under "Experimental Procedures" with varying concentrations of the shown inhibitors. The data are shown as the mean ± S.E. (n = 3-5) except for PAP1, which is in singlicate due to the limited availability of this compound.

    DISCUSSION
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Procedures
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Discussion
References

The inhibition of ceramide synthase by FB1 and other fumonisins has been attributed, in large part, to similarities between the AP1 and the sphingoid base substrates for this enzyme (1, 7). Nonetheless, this analogy is not fully convincing because AP1 differs from natural sphingoid bases in ways that are often important to enzyme-ligand interactions, namely the stereochemistry in the vicinity of the group to be modified (i.e. the 2-amino and 3-hydroxyl groups are threo for fumonisins and erythro for sphingoid bases), the absence of a hydroxyl group at position 1 (which might be important in binding, or orientation of the amino group for acylation), and the presence of an additional hydroxyl at position 5 (which might impose bulk or polarity in a key region of the enzyme active site). The structure-function analyses presented in this report alleviate these concerns by establishing that both erythro- and threo- 1-deoxy, 5-hydroxy-sphinganines inhibit ceramide synthase and serve as reasonably good substrates.

These findings extend the recent evaluation of the stereoisomers of sphinganine and sphingosine as substrates for ceramide synthase (25), which ranked the stereoisomers (based on the apparent Vmax/Km): D-erythro-(2S,3R) > D-threo-(2R,3R) L-threo-(2S,3S) > L-erythro-(2R,3S). The Vmax/Km only differed by approximately 20-fold for naturally occurring D-erythro-sphinganine versus L-threo-sphinganine4 (Fig. 3) and arose from a 5-fold higher Vmax and a 4-fold lower Km. These results also agree with a much earlier finding by Stoffel and Bister (26) that radiolabeled ceramides can be found in rat liver after intravenous injection of any of the radiolabeled stereoisomers of sphinganine. The reason(s) for this tolerance of multiple structural modifications are not known,5 but a less restricted binding site might allow ceramide synthase(s) to accommodate the many different types of sphingoid bases that are found in nature (27). Because of this low degree of specificity, however, future studies should explore whether ceramide synthase can acylate other hydrophobic amines, including pharmaceuticals and toxins.

Compounds of particular interest are AP1 and FB1, which are known inhibitors of ceramide synthase (13) but have not been analyzed as possible substrates. The acylation of AP1 was similar to that for the threo-analogs; however, FB1 was not detectably acylated, which is consistent with our suggestion (13, 20) that the tricarballylic acid side chains of FB1 occupy the acyl-CoA binding site and thereby block access of this co-substrate.

A somewhat unexpected finding of this study was that PAP1 is highly cytotoxic, apparently as an inhibitor of ceramide synthase. This inhibition by N-acyl,1-deoxysphinganines represents a new category of ceramide synthase inhibitor. Thus, PAP1 causes an increase in cellular sphinganine, which is cytotoxic for many cell types (29-33)6 and has been shown to mediate the toxicity of FB1 for HT29 cells (19). Although PAP1 might also be toxic as a ceramide analog because ceramides can induce apoptosis, this probably does not play a role in the toxicity of PAP1 because the effects of N-acylsphingosines (ceramides) are usually stereoselective and require the 4,5-trans-double bond of the sphingosine backbone (28).

Feeding AP1 to rats causes lesions in liver and kidney that are indistinguishable from those caused by FB1 (21), and the organ-specific effects of feeding nixtamalized Fusarium moniliforme culture material (which contains AP1) to rats are similar to those of the diet prepared from untreated (FB1-containing) culture material (21). AP1 also appears to have the same liver cancer promoting activity as FB1 (34). Heretofore, these in vivo effects of AP1 have been somewhat puzzling because AP1 is less potent than FB1 as an inhibitor of ceramide synthase in vitro (13); however, if AP1 is converted to an even more potent metabolite (PAP1), this could account for this discrepancy. Further evidence for a similar mechanism of action of FB1 and AP1 is the close correlation between the toxicity of FB1 or AP1 for rats and elevations in sphinganine (22).

The toxicity of FB1, AP1 (19), and PAP1 for HT29 cells, an intestinal cell line, implies that consumption of fumonisins may affect the gastrointestinal tract since a substantial portion of an administered dose is found in intestinal epithelial cells (35), excreta, bile, and gut contents as FB1, partially hydrolyzed FB1 and AP1 (36). Little is known about the intestinal uptake and fate of AP1; however, sphingoid bases are rapidly taken up by intestinal cells (37); therefore, it is likely that AP1 is taken up and converted to PAP1. This scenario might explain the recent report that human consumption of moldy maize (containing fumonisins) resulted in abdominal pain, borborygmi, and diarrhea (38).

    ACKNOWLEDGEMENTS

The authors thank M. Herderich for assistance with some of the MS analyses, R. T. Riley for helpful discussions, and Winnie Scherer for assistance in preparing the manuscript.

    FOOTNOTES

* This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (HU 730/1-2), the Josef Schormüller Gedächtnisstiftung, Berlin, National Institutes of Health Grant GM 46368, and a Focused Giving Award from Johnson and Johnson.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed. Tel.: 404-727-5978; Fax: 404-727-3954; E-mail: amerril{at}emory.edu.

1 The abbreviations used are: FB1, fumonisin B1; AP1, aminopentol; PAP1, N-palmitoyl AP1; FAB-MS, fast atom bombardment mass spectroscopy; ES-MS, electrospray mass spectroscopy; HPLC, high pressure liquid chromatography.

2 For more information about these syntheses, consult the doctoral thesis of David S. Menaldino (UMI order no. AAI9328152; print index reference DAI 54-07B:3626) or contact Dr. D. C. Liotta.

3 To determine whether the presence of the carrier solvents (ethanol:dodecane, 98:2) might have enhanced the toxicity of PAP1, this solvent mixture was also added to cells incubated with FB1. Under these conditions, 50 µM FB1 reduced the cell number by 37 ± 2%, which was not greater than the reduction by FB1 alone.

4 This should be borne in mind when L-threo-sphinganine is added to cells to inhibit sphingosine kinase because this treatment will lead to formation of the L-threo-(dihydro)ceramides, which may disrupt other aspects of sphingolipid metabolism and signaling.

5 More detailed mechanistic studies are not possible until this enzyme has been purified and can be characterized under more defined conditions.

6 This includes AP1 (19) and synthetic analogs of AP1 (29), but these earlier studies did not evaluate the mechanism for the toxicity, nor the formation of the N-acyl derivatives.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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