The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 214-220

Fumonisin B₁ Consumption by Rats Causes Reversible, Dose-Dependent Increases in Urinary Sphinganine and Sphingosine^1,2

Elaine Wang^*, Ronald T. Riley, Filmore I. Meredith, and Alfred H. Merrill Jr.^{*, 3}

^* Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA and  U. S. Department of Agriculture, Agriculture Research Service, Toxicology and Mycotoxins Research Unit, Athens, GA 30604-5677, USA

	ABSTRACT

Abstract Introduction Methods Results Discussion References

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 µg 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 µg 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 µg FB₁/g for up to 60 d. When rats were fed FB₁ (10 µg 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 µg FB₁/g for 10 d, then changed to 1 µg FB₁/g, the amounts of sphingoid bases in urine were the same as for rats that were continuously fed 10 µg 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 µg FB₁/g (which does not, alone, alter urinary sphingoid bases) will sustain the elevation caused by previous exposure to 10 µg FB₁/g raises the possibility that even low levels of fumonisins could be deleterious when an animal is occasionally exposed to higher amounts.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Fumonisins are a group of structurally related mycotoxins produced by Fusarium moniliforme (Sheldon) (Bezuidenhout et al. 1988), F. proliferatum (Ross et al. 1990) and related fungi (Nelson et al. 1992, Thiel et al. 1991) that are prevalent on corn, sorghum and other grains throughout the world (Marasas et al. 1984). Fumonisins have diverse effects on animals and, depending on the species, cause neurotoxicity, hepatotoxicity, nephrotoxicity, immunosuppression (and sometimes immunostimulation), developmental abnormalities, liver tumors and other disorders (Marasas 1996, Riley et al. 1996). They are of major agricultural concern as the cause of equine leukoencephalomalacia (Marasas et al. 1988) and porcine pulmonary edema (Harrison et al. 1990). The role of fumonisins in human health is unclear; however, consumption of F. moniliforme-contaminated corn has been associated with human esophageal cancer in areas of southern Africa, China and other countries (reviewed in Marasas 1996).

A major cellular target of fumonisins is the enzyme ceramide synthase (sphingosine [sphinganine] N-acyltransferase), which is potently inhibited by fumonisins of the "B" series (Merrill et al. 1993, Norred et al. 1992, Wang et al. 1991). Inhibition of this enzyme in intact cells causes accumulation of its substrate sphinganine from the de novo biosynthesis of sphingolipids, and under some conditions (and to a lesser extent), sphingosine from sphingolipid turnover, as depicted in Figure 1 (Merrill et al. 1993). Upon cellular accumulation of sphinganine (and sphingosine), these compounds leave the cell (as also depicted in Fig. 1) and appear in blood (Wang et al. 1992) and urine (Riley et al. 1994a); therefore, the appearance of elevated sphingoid bases appears to be a good biomarker for exposure to this mycotoxin (Riley et al. 1993 & Riley et al. 1994b).

View larger version (33K): [in this window] [in a new window]   Fig 1. Schematic representation of the inhibition of ceramide synthase by fumonisin B1, with accumulation of cellular sphinganine and sphingosine as well as release of these compounds from the cells to plasma or urine.

Much attention has been focused on the effects of fumonisins on brain, lung, and liver, although the kidney is also sensitive to this mycotoxin based on studies of renal cells in culture (Yoo et al. 1992 and Wang et al. 1996) and animals given fumonisins (Voss et al. 1989, Voss et al. 1993, Riley et al. 1994a, Suzuki et al. 1995, Bondy et al. 1996, Voss et al. 1996, LaBorde et al. 1997, de Nijs 1998). Elevations in sphingoid bases can be detected in urine (Riley et al. 1994a); therefore, this study characterized the dose response, time course and reversibility of the changes in urinary long-chain bases upon feeding fumonisin B₁ (FB₁),⁴ the most prevalent fumonisin in most food (Marasas 1996).

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  The FB₁ was isolated from culture material by the procedure of Meredith et al. (1996) and was >70% pure based on comparison of nmol determined by high-performance liquid chromatography to weight (a substantial portion of the difference is probably due to salts). The fumonisin aminopentol (AP₁) was synthesized from FB₁ by basic hydrolysis, as described by Wilson et al. (1990), and was the only compound detected by high-performance liquid chromatography. The C₂₀-sphinganine was synthesized as described by Nimkar et al. (1988) as part of other studies by Merrill and coworkers (under NIH grant GM 46368). The powdered AIN-76A diet (American Institute of Nutrition, 1977) was purchased from PMI Feeds (Richmond, IN). Other chemicals were obtained from commercial suppliers.

Feeding protocols.  Each experiment used three to six male, Sprague-Dawley derived rats (Harlan, Indianapolis, IN) that weighed approximately 50 to 74 g at the start of the feeding studies. Prior to feeding the experimental diets, the rats were maintained on a powdered AIN-76 diet for 1 wk. Thereafter, they were placed in metabolic cages (that separate urine and feces) and fed the same diet containing 0 to 50 µg of FB₁/g diet (the FB₁ was carefully mixed with the diets to ensure uniform distribution) for the times described in the Results section. Urine was collected daily and stored at 20°C until analysis. Upon completion of the feeding, the rats were anesthetized with ether, and blood, liver and kidney were collected. Tissues were stored at 80°C for later analyses.

The animal care and use protocol was approved by the National Veterinary Services Laboratories Animal Care and Use Committee. Experiments were conducted in accordance with the "Guide for Care and Use of Laboratory Animals," NIH publication No. 88-23, 1985.

Analyses of long-chain bases.  The amounts of total sphingosine and sphinganine in tissues (liver and kidney) and serum were determined as described by Merrill et al. (1988) and Wang et al. (1991), respectively. The analyses of sphingolipids in urine were conducted with 1 mL aliquots as follows: (1) 1 mL of methanol and 288 pmol of C₂₀-sphinganine (as an internal standard) were added, followed by incubation for 1 h at 37°C; (2) 15 µL of concentrated NH₄OH were added, followed by 0.3 mL of CHCl₃ and incubation for 30 min at 37°C; (3) 1.7 mL of CHCl₃ (for a CHCl₃/methanol ratio of 2:1) and 2 mL of H₂O were added, followed by gentle mixing and separation of the two phases by centrifugation; (4) the upper phase was discarded and the CHCl₃ phase washed with several mL of H₂O (twice); after removal of the water, the CHCl₃ extract was further dried by passage through a small column (e.g., made from a Pasteur pipet) containing anhydrous crystalline Na₂SO₄; (5) the CHCl₃ was evaporated using a Speed Vac Concentrator (Savant, Farmingdale, NY) with care to avoid overheating); (6) the residue was resuspended in 1 mL of 0.1 mol/L KOH in CHCl₃/methanol (1:4, v/v), and incubated for 1 h at 37°C; (7) the samples were then cooled to room temperature, 1 mL each of CHCl₃ and H₂O were added, the free long-chain bases were recovered in the CHCl₃ phase (and dried over Na₂SO₄), the solvent was removed in vacuo; and, (8) the lipids were dissolved in 500 µL of mobile phase (91% methanol/9% 5 mmol/L potassium phosphate buffer, pH 7.4, v/v) and derivatized with 100 µL of 0.05% (w/v) ortho-phthaldialdehyde in 3% borate, pH 10.5 (w/v). Aliquots were injected onto a C18-reversed-phase high-performance liquid chromatography column (Waters Chromatography, Milford, MA) and eluted isocratically with mobile phase as described by Merrill et al. (1988). The amounts of sphinganine and sphingosine were calculated using the relative areas of the unknowns vs. the C₂₀-sphinganine internal standard.

Statistical analyses.  Each experimental group consisted of three to six rats, and each sample was analyzed in triplicate. Comparisons among groups were evaluated for statistical significance by analysis of variance using Instat® software (Instat, San Diego, CA), followed by pair-wise comparisons to derive the probability values for the differences between specific groups or time points for a given group. Differences were considered significant at P < 0.05. Values are means ± SEM.

	RESULTS

Abstract Introduction Methods Results Discussion References

Time-course and dose-response changes in sphinganine in urine.  The amounts of sphinganine in urine remained constant for the rats fed no FB₁ over this time course (Fig. 2). Using the means from all of the time points shown, the amount of sphinganine in urine from rats fed no FB₁ was 0.28 ± 0.03 µmol/L (n = 20).⁵ Further analyses were conducted approximately every third day for a total of 63 d (data not shown), and the amount of sphinganine was also constant over this longer period. The mean for all of the analyses of the control rats (n = 35 d of analysis) was 0.30 ± 0.02 µmol sphinganine/L.

View larger version (25K): [in this window] [in a new window]   Fig 2. Time course of the changes in sphinganine in urine of rats fed an AIN-76A diet supplemented with varying amounts of fumonisin B1 (FB1). Points represent the mean ± SE of urine from three rats. After d 5 to 7 (see text) means for the rats fed 5 mg FB1/g diet were significantly different (P < 0.05) from the controls that were not fed fumonisin. Due to the scale of the figure, it is not possible to see the variance for the rats fed 0 µg/g diet FB1, which was approximately half of the mean at each time point.

Rats that consumed 1 µg FB₁/g diet had the same amounts of urinary sphinganine as the no-FB₁ controls. However, after a 5 to 7 d lag, rats fed 5, 10 or 50 µg FB₁/g diet had significantly higher amounts of sphinganine in urine (Fig. 2). The "lag" is not an artifact of the scale of the figure. For example, the sphinganine amount on d 4 for the rats fed 50 µg FB₁/g was 0.41 ± 0.07 µmol/L (n = 5), which was not significantly different from the control; whereas, on d 5, the amount of sphinganine for rats fed 50 µg FB₁/g was 1.20 ± 0.09 µmol/L.

Considerable variability existed in the amounts of sphinganine in urine of the rats fed 5 to 50 µg FB₁/g diet, as might be expected since the sphinganine is present mainly in the urinary sediment (due to its association with cells and membrane fragments) (Riley et al. 1994a). After the lag period, the amounts of sphinganine in urine in these groups ranged from ca. 3 to 10 µmol of sphinganine/L, which represents a ca. 10- to 30-fold elevation over the control.

Because urinary sphinganine was not elevated at any time point for the rats fed 1 µg FB₁/g diet, urine from this group was analyzed after an additional 40 d of feeding, which still failed to reveal an increase in sphinganine over the controls (data not shown).

Changes in urinary sphingosine.  The amounts of sphingosine in urine were more variable over time (Fig. 3), as has been seen in previous analyses (Riley et al. 1994a, Wang et al. 1992). However, the mean sphingosine concentration in the control rats over the entire time course was 0.47 ± 0.06 µmol/L (n = 20). Therefore, rat urine usually contains somewhat greater amounts of sphingosine than sphinganine.

View larger version (32K): [in this window] [in a new window]   Fig 3. Time course of the changes in sphingosine in urine of rats fed an AIN-76A diet supplemented with varying amounts of fumonisin B1 (FB1). Points represent the mean ± SE of urine from three rats.

Due to day-to-day variability in the amounts of sphingosine in urine, comparisons among the groups are difficult. However, when the d 10 to 20 data for all of the rats in each group were combined (n = 30), urinary spingosine in rats fed 0 (control), 1, 5, 10 and 50 µg FB₁/g diet were: 0.45 ± 0.09, 0.57 ± 0.06, 1.29 ± 0.11 (P < 0.001 vs. control), 1.12 ± 0.11 (P < 0.001), and 0.80 ± 0.04 (P < 0.05) µmol/L urine, respectively. Therefore, FB₁ significantly increased the urine sphingosine concentration when fed at 5, 10 or 50 µg/g diet, but not at 1 µg/g (Fig. 3). The highest amount of sphingosine (1.9 ± 0.35 µmol/L) was detected on d 11 in rats fed 10 µg FB₁/g, and represents only a 3-fold elevation in sphingosine.

Changes in the sphinganine-to-sphingosine ratio.  Some of the variability in sphingosine and sphinganine is probably due to the difficulty of extracting and analyzing small amounts of sphingolipids (Merrill et al. 1988). Losses during extraction can be estimated using C₂₀-sphinganine as an internal standard (Merrill et al. 1988, Riley et al. 1994b); nonetheless, an internal standard that is added at the time of extraction will not correct for all losses, such as those that occur during storage. An alternative way to estimate changes in sphinganine is to determine the sphinganine-to-sphingosine ratio (Merrill et al. 1988, Riley et al. 1994b). The results using this ratio (Fig. 4) generally mirrored the changes in sphinganine (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig 4. Time course of the changes in the sphinganine-to-sphingosine ratio in urine from rats fed an AIN-76A diet supplemented with varying amounts of fumonisin B1 (FB1). The data are from Figures 2 and 3. After d 5 to 7 (see text) all of the results for the rats fed 5 mg FB1/g diet were significantly different (P < 0.05) from the controls that were not fed fumonisin.

Changes in sphinganine and sphingosine in liver, kidney and plasma.  Consistent with the elevation of sphinganine in urine, the kidney showed significant (i.e., 90- to 160-fold) elevation in sphinganine after 10 and 21 d of feeding 50 µg FB₁/g diet. Sphinganine and sphingosine were increased in liver after 10 d, but by no more than 86% (Table 1). Plasma sphingolipids were elevated in rats fed FB₁ on d 21 (P < 0.05; Table 1).

Although urinary sphingoid bases were not elevated significantly by feeding FB₁ at 1 µg/g diet for 40 d, analyses of kidneys revealed 2-fold greater free sphinganine (5.1 ± 0.7 vs. 1.7 ± 0.2 nmol/g for the FB₁-treated rats vs. the controls, P < 0.01) and almost 100% greater free sphingosine (20.3 ± 1.7 vs. 11.2 ± 0.9 nmol/g for the FB₁-treated rats vs. the controls, P < 0.01). These small differences indicate that even this low amount of FB₁ affects sphingolipid metabolism in kidney.

Reversibility of the elevation of sphinganine in urine.  The amounts of sphinganine in urine returned to control levels within 10 d after switching rats from diets containing 10 µg FB₁/g to 0 µg FB₁/g (Fig. 5). However, when they were switched from 10 µg FB₁/g to 1 µg FB₁/g, sphinganine remained elevated, and the amounts were comparable to that of rats fed 10 µg FB₁/g for the entire time course. A similar pattern is seen when the data are expressed as the sphinganine-to-sphingosine ratio (Fig. 6). The elevated sphinganine was not due to longer feeding of 1 µg FB₁/g because no elevation was seen when rats were fed this amount of FB₁ for the entire period. Therefore, the level of sphinganine that is found when rats are given a low level of FB₁ (1 µg/g) can be affected by their prior exposure to a higher amount.

View larger version (23K): [in this window] [in a new window]   Fig 5. Time course of the changes in urinary sphinganine for rats that were fed 10 µg FB1/g diet for 10 d, followed by a change of the diet to 0 (closed circles) or 1 (squares) µg FB1/g diet. The data are shown as the mean ± se, n = 6; the asterisks indicate the timepoints where the results with 1 and 0 µg FB1/g diet were significantly different (P < 0.001).

View larger version (20K): [in this window] [in a new window]   Fig 6. Time course of the sphinganine-to-sphingosine ratio of urine from rats fed 10 µg FB1/g diet for 10 d followed by a change to 0 or 1 µg FB1/g diet. The asterisks indicate the timepoints where the results with 1 and 0 µg FB1/g diet were significantly different (P < 0.001).

Effects of feeding FB₁ in a nonpurified, natural composition diet.  The AIN-76A diet used in these studies is composed of defined ingredients (casein, corn oil, vitamins, etc.) that are essentially free of FB₁; however, diets composed of natural ingredients (which often includes corn) are variably contaminated with FB₁ up to ca. 3 µg/g diet, and the level of contamination has been correlated with the sphinganine-to-sphingosine ratio (Merrill et al. 1996b). Figure 7 presents a time course for the ratio in urine from rats fed a typical, commercial, natural composition diet vs. the same diet containing 50 µg FB₁/g diet. The ratio for the rats fed the natural composition diet was highly erratic,⁶ but was consistently >1, which rarely occurs in rats fed the AIN-76 diet (Fig. 4 and data not shown). Addition of 50 µg of FB₁/g diet increased the sphinganine-to-sphingosine ratio to ca. 4 within 5 d, and by d 10 the ratio was 5 to 6 (Fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig 7. Time course changes in the sphinganine-to-sphingosine ratio in urine from rats fed an AIN-76A diet, a standard laboratory nonpurified diet, or that diet plus 50 µg FB1/g diet. The asterisks indicate the timepoints where the results with the nonpurified diet and the nonpurified diet plus 50 µg FB1/g diet were significantly different (P < 0.01).

Effects of feeding the aminopentol (AP₁) of FB₁.  The processing of corn for some foods, such as tortillas, involves a process termed "nixtamalization," which converts a substantial portion of the FB₁ to an AP₁ that lacks the tricarballylic acid sidechains (Hendrich et al. 1993, Voss et al. 1996). When rats were fed 50 µg of AP₁/g diet (in an AIN-76A diet), there was no increase in the tissue sphinganine or sphingosine, respectively, in kidney (2.0 ± 0.2 nmol/g; 13.8 ± 2.3 nmol/g) or liver (0.7 ± 0.1 nmol/g; 6.8 ± 0.7 nmol/g), or the concentration in plasma (0.07 ± 0.05 µmol/L; 0.21 ± 0.12 µmol/L) as compared to the control levels shown in Table 1.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Although fumonisins are toxic for liver, lung and brain, kidneys have also been affected in numerous species, including pigs and mice (Harvey et al. 1996, Tsunoda et al. 1998), and appear to be the most sensitive organs in male rats (Bondy et al. 1996, Riley et al. 1994a) and rabbits (LaBorde et al. 1997). Voss et al. (1989, Voss et al. 1993, Voss et al. 1995) first reported changes in renal morphology, and characterized the ultrastructural lesions as swelling and dilation of mitochondrial cristae, an increase in the electron dense vacuoles (which sometimes contain lamellar membranous whorls) and disruption of the normal architecture of the deep infoldings of the basal region of the epithelial cells in kidneys from male Sprague-Dawley rats fed dietary fumonisin B₁ at 15 µg/g diet (Riley et al. 1994a). Suzuki et al. (1995) reported disseminated single-cell necrosis in proximal tubules accompanied by focal-accentuated tubular degeneration leading to desquamation of epithelial cells into the tubular lumina in male Sprague-Dawley rats administered FB₁ intraperitoneally (7.5 mg/kg body weight for 4 d). In addition, tubular epithelial apoptosis was seen in the inner cortex (nuclear condensation, cytoplasmic eosinophilia and degenerative changes in the tubular epithelium) (deNijs 1998, Tolleson et al. 1996).

Kidney function was shown to be impaired in male Sprague-Dawley rats at the lowest FB₁-dose tested (1 mg/kg body weight, administered by gavage for 11 d), which is roughly equivalent to consumption of 12.5 µg FB₁/g diet (Bondy et al. 1995 and Bondy et al. 1996). Fumonisin administration decreased urine osmolality, which reflects the loss of urine-concentrating ability, and increased loss of protein into urine, as reflected in total protein and elevated activities of lactate dehydrogenase, -glutamyltranspeptidase and N-acetyl--D-glucosaminidase. Decreased organic ion uptake was shown using renal cortical slices from male rats exposed to FB₁ (Bondy et al. 1995 and Bondy et al. 1996).

For both cells in culture and kidneys from animals treated with FB₁, the earliest biochemical change that has been noted is an elevation of sphinganine due to inhibition of ceramide synthase (Riley et al. 1994a, Yoo et al. 1992). However, it is not possible to conclude that the sphingoid bases in urine are solely derived from de novo sphingolipid biosynthesis by the kidney because many cell types were affected by FB₁ (Merrill et al. 1996a, Riley et al. 1996) and most, if not all, tissues appear to have the capacity for long-chain biosynthesis de novo (Merrill et al. 1985). Nonetheless, renal cells are active in sphingolipid biosynthesis, remodeling and signaling (Shayman 1996), and it is likely that at least a portion of the sphingoid bases that accumulate in urine of fumonisin-treated animals is produced in the kidney.

In this study, sphinganine increased over 100-fold in kidney within 10 d of feeding FB₁ (the first time point at which animals were killed for analyses of tissue sphingolipids), and urinary sphinganine was elevated ca. 15-fold by d 5 to 7- and 30-fold on d 10. It is somewhat puzzling that feeding 50 µg AP₁/g diet did not affect the amounts of sphinganine in kidney (or liver or plasma) because even though AP₁ is less potent than FB₁ as an inhibitor of ceramide synthase (Merrill et al. 1993, Norred et al. 1997), recent studies with HT29 cells in culture (Humpf et al. 1998) showed that AP₁ can be acylated to N-palmitoyl-AP₁, which is a more potent inhibitor of ceramide synthase. This warrants further study because AP₁ is toxic (Hendrich et al. 1993, Voss et al. 1996), and the mechanism for this toxicity is less well characterized than for FB₁.

Measurement of sphingoid bases provides not only a bimarker for disruption of sphingolipid metabolism by FB₁, but also, the amounts of a key mediator of the toxicity of this mycotoxin. Increases in free sphinganine in kidney and/or liver in rats and mice given fumonisins were correlated with the severity of the lesions (de Nijs 1998, Riley et al. 1994a, Tsunoda et al. 1998, Voss et al. 1998). Renal cells in culture (Wang et al. 1996, Yoo et al. 1992) also exhibit growth arrest, morphological changes and cell death (via apoptosis) when treated with FB₁. Yoo et al. (1992, Yoo et al. 1996) showed that FB₁ causes sphinganine accumulation and growth arrest/cytotoxicity in LLC-PK₁ cells; moreover, they established a cause-and-effect relationship between these responses because the initial growth arrest and cytotoxicity were reversed by suppression of sphinganine accumulation using an inhibitor of an upstream enzyme of the pathway (Yoo et al. 1996). As participants in cellular signal transduction pathways (Spiegel and Merrill 1996), there are many possible mechanisms whereby sphingoid bases are growth inhibitory and cytotoxic, including the inhibition or activation of cellular protein kinases (including protein kinase C) (Stevens et al. 1990), ion transporters and other signal transduction pathways (for reviews see Merrill et al. 1996b, Merrill et al. 1996c). In our opinion, the cellular effects of sphingoid bases that are most likely to be relevant to the nephrotoxicity of FB₁ are the induction of retinoblastoma protein dephosphorylation (Pushkareva et al. 1995, Ciacci-Zanella et al. 1998), and the activation of apoptosis (Ohta et al. 1995, Sakakura et al. 1996, Sweeney et al. 1996, Schmelz et al. 1998).

This study has uncovered several questions for further investigation. First, do other components of the diet affect the response to fumonisins? Second, do amounts of FB₁ that are not toxic when consumed alone become more damaging if there is occasional consumption of higher amounts, since prior exposure to a high level of FB₁ (e.g., 10 µg/g) had such a profound effect on the levels of sphingoid bases in urine upon subsequent consumption of a lower amount (e.g., 1 µg/g) (Figs. 5 and 6)? This scenario of exposure may be common because mycotoxins are usually not distributed evenly in food. For comparison, the mean combined fumonisin level of commercial corn harvested in Iowa, Wisconsin and Illinois was 3.4 to 4.5 µg/g between 1988 and 1991 (Murphy et al. 1993). And third, can these biomarkers be used to explore the relative sensitivity of humans to fumonisins (sphingoid bases can also be detected in human urine) (Solfrizzo et al. 1997)? Little is known about the effects of fumonisins on humans, although it is possible that the kidneys will be affected since sphingosine was shown to be toxic for a human proximal tubular cell line (HK-2 cells) (Iwata et al. 1995), and sphinganine is elevated in urine from nonhuman primates fed fumonisins (Shephard et al. 1996). Short-term studies of urinary sphingoid bases in populations that are naturally exposed to this mycotoxin would help clarify this question, especially if conducted in conjunction with markers of renal function.

	FOOTNOTES

Manuscript received 17 February 1998. Initial reviews completed 15 May 1998. Revision accepted 26 October 1998.

	ACKNOWLEDGMENT

We thank Christopher Alexander and Dennis Liotta for the C₂₀-sphinganine, and Winnie Scherer for assistance in preparing the manuscript for publication.

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