Persistence and Reversibility of the Elevation in Free Sphingoid Bases Induced by Fumonisin Inhibition of Ceramide Synthase

E. N. Enongene*, R. P. Sharma{dagger}, N. Bhandari{dagger}, J. D. Miller{ddagger}, F. I. Meredith*, K. A. Voss* and R. T. Riley*,1

* Toxicology and Mycotoxin Research Unit, R. B. Russell Research Center, USDA/ARS, P.O. Box 5677, Athens, Georgia 30604-5677; {dagger} College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602; and {ddagger} Department of Chemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6

Received November 29, 2001; accepted February 1, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 B1 (FB1), (2) the minimum threshold dose of FB1 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 FB1, 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 FB1/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 FB1 followed by daily exposure at low levels will prolong the elevation of sphinganine in kidney. In cultured renal cells FB1 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.

Key Words: fumonisin; fusarium verticillioides; sphingolipids; liver; kidney; gastrointestinal; ceramide synthase; serine palmitoyltransferase; ISP-1; LLC-PK1 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fumonisins (FB) are mycotoxins produced by the fungus Fusarium verticillioides (synonymous with F. moniliforme), a common pathogen in corn (Shephard et al., 1996Go). They are known to be potent inhibitors of ceramide synthase (sphinganine [sphingosine] N-acyltransferase; Wang et al., 1991Go). FB inhibition of ceramide synthase causes a rapid increase in free sphinganine, the immediate sphingoid base precursor in the de novo biosynthesis of dihydroceramide and ceramide (Fig. 1Go). High levels of free sphinganine (and sometimes sphingosine) in tissues and biological fluids are used as a biomarker for FB exposure (for review see Marasas et al., 2000Go). A recent study has reported an increased ratio of free sphinganine to free sphingosine in urine of humans consuming FB contaminated corn (Qiu et al., 2001). The liver and kidney are target organs for ceramide synthase inhibition in all animal species tested thus far.



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FIG. 1. Outline of the de novo sphingolipid biosynthetic pathway and the sphingolipid turnover pathway in an animal cell and inhibition of ceramide synthase by FB1 and serine palmitoyltransferase by ISP-1.

 
In mice dosed subcutaneously with FB the increase in free sphinganine in kidney is much more persistent than in either liver or the intestinal epithelia (Enongene et al., 2000Go). In liver the free sphinganine concentration returned rapidly to the control level suggesting that liver handled either fumonisins or sphingoid bases quite differently than kidney. The persistent elevation of free sphinganine has also been reported in cultured pig renal epithelial cells (Riley et al., 1998Go).

The persistence of free sphinganine elevation in kidney may be a consequence of a combination of persistent inhibition of ceramide synthase and slow degradation or elimination of free sphinganine. In a study using [14C]FB1 in rats, Norred et al. (1993) reported that a small amount of radioactivity was retained in both liver and kidney long after the FB1 was eliminated from blood. It was hypothesized that the retained material might be that which is bound to ceramide synthase (Norred et al., 1993Go). A similar persistent retention of trace amounts of [14C]FB1 has been seen in cultured renal epithelial cells (Riley et al., 1998Go). The ability of low doses of FB1 to prolong the effects of ceramide synthase inhibition in kidney was suggested by Wang et al. (1999) who found that maintaining rats on a low FB1 diet (1 ppm in AIN 76 diet), which by itself was insufficient to cause an increase in free sphinganine in rat urine, prolonged the elevation of urinary free sphinganine that had been induced by feeding a diet containing 10 ppm FB1. Thus, while ceramide synthase inhibition is reversible, the time required for reversal of the effects of inhibition may be prolonged by noninhibitory doses (Merrill et al., 2001Go). A similar persistence was seen in cultured pig kidney epithelial cells (LLC-PK1) where free sphingoid bases remained significantly elevated for at least 72 h after FB1 was removed from the culture medium (Riley et al., 1998Go). The prolonged elevation of free sphingoid bases in some cell types could be due to a combination of persistent inhibition of ceramide synthase and differences in the kinetics of sphinganine biosynthesis (serine palmitoyltransferase activity) and degradation (via sphinganine kinase).

The purpose of this study was to determine the following: (1) the time course for sphingoid base elevation in the small intestines, liver, and kidney following a single oral dose of FB1 (25 mg/kg body weight [bw]), (2) the minimum threshold FB1 dose that would sustain the free sphingoid base concentration in kidney (the tissue most sensitive to accumulation of free sphinganine) following a single oral dose of 25 mg FB1/kg bw, and (3) the importance of the balance between the rate of sphingoid base biosynthesis and degradation in the persistence of FB1-induced free sphingoid base accumulation using cultured pig kidney epithelial cells (LLC-PK1) and the serine palmitoyltransferase inhibitor, ISP-1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies in Vivo
Animals.
Eight-week-old male Swiss NIH mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and acclimatized for 1 week at the USDA animal facility, Athens, GA. The animals were housed in groups of 4 each, provided free access to FB1-free (< 0.4 ppm) commercial feed (22/5 rodent diet, from Harlan-Teklad, Madison, WI), and water. They were maintained in a controlled temperature (22°C) and humidity (45%) environment with a 12-h light/dark cycle. Food, water intake, and body weight were recorded daily. Protocols for animal use were approved by the Russell Research Center Institutional Animal Care and Use Committee.

Animal studies.
Apart from the zero time points (water control group), all other mice were administered doses of FB1 dissolved in sterile water (10 ml/kg bw) by gavage using stainless steel ball-tipped needles. Three experiments were conducted each following slightly different protocols.

Experiment 1.
The mice received a single dose (25 mg/kg bw) of FB1 and tissues (liver, kidney, and epithelial cells from the small intestines) were collected at 0, 2, 4, 8, 12, 24, and 48 h post dosing (n = 3 to 7/time point). Animals were killed by decapitation. The dose was selected based on our previous studies (Enongene et al., 2000Go), which showed that 25 mg/kg bw did not produce overt toxicity after 48 h. Details of effects on cytokine expression associated with exposure to a single 25 mg/kg bw po dose of FB1 have been described elsewhere (Bhandari et al., in press). Kidney and liver samples were immediately frozen on dry ice, and stored at –80°C until analyzed for sphingolipids as described below. Epithelial cells were collected from the small intestines using a modification of the method of Weiser (1973). Briefly, a portion of the GI tract (duodenum to caecum) was excised, and undigested materials were removed by flushing the lumen with 30 ml of 0.154 M NaCl, containing 1 mM dithiothreitol (DTT) at room temperature using a stainless steel cannula affixed to a syringe. The washed intestines were ligated at one end with fine thread, filled with solution A (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8mM KH2PO4, 5.6 mM Na2HPO4, pH 7.3), ligated at the other end and incubated at 37°C for 15 min. One end was snipped off to discard solution A. The lumen was then filled with solution B (Hanks buffered saline without calcium or magnesium, 1.5 mM EDTA, 0.5 mM DTT, pH 7.3); the segment was ligated and then incubated for 1 h at 37°C. After 1 h, one end was removed and solution B collected in a 15 ml conical tube. The other end of the segment was also removed and the lumen washed with about 10 ml of solution B to remove any remaining cells. The tubes containing the detached cells (combined solutions B) were centrifuged at 900 x g at 4°C for 10 min, supernatant aspirated and cell pellets frozen at –80°C until analyzed for sphingolipids.

Experiment 2.
The mice received a single po dose (25 mg/kg bw) of FB1. Kidneys were collected over a 120 h time-course (0, 6, 12, 24, 48, 72, 96, and 120 h, n = 3 to 4/time point) and stored as described for mice in Experiment 1.

Experiment 3.
The mice received either a single po dose of 25 mg/kg bw or 0.03 mg/kg bw of FB1 followed by a daily po dose of either 0.03 mg FB1/kg bw or 0 mg FB1/kg bw (water) for 4 more days (n = 6 to 8/group). A control group was given water only for 5 consecutive days. Kidneys were collected 24 h after the last dose (120 h). The 0.03 mg/kg bw dose of FB1 was selected based on a preliminary experiment that showed that a single dose of 0.3 mg/kg bw, but not 0.03 mg/kg bw, caused an elevation in free sphinganine in kidney.

Studies in Vitro
Cell culture.
Pig kidney epithelial cells (LLC-PK1, CRL 1392, passage 197) were obtained from the American Type Culture Collection (Rockville, MD) and maintained as described previously (Yoo et al., 1992Go). Briefly, cells were grown and maintained in 25-cm2 culture flasks containing DMEM/Ham's F12 (1:1) with 5% fetal calf serum at 37°C and 5% CO2. For all experiments cells were subcultured at approximately 15,000 to 30,000 viable cells/cm2 in 10-cm2 dishes. Cells were allowed to attach and grow to at least 90% confluence (3 to 5 days) prior to addition of FB1 or ISP-1.

Studies in LLC-PK1 cells.
The uptake and elimination of [U-14C]FB1 and effects of FB1 on accumulation and persistence of free sphingoid bases in cells was determined using confluent cultures of pig kidney LLC-PK1 cells cultured as described above. Stock solutions of [U-14C]FB1 were dissolved in phosphate buffered saline (PBS). Cell were grown in 10 cm2 dishes for 5 days and then the growth medium was removed and replaced with PBS plus 10 mM glucose (PBSG) containing various concentrations of [U-14C]FB1 (0 to 1000 µM FB1). Preliminary experiments found that FB1 accumulation under these conditions was linear for at least 2 h. Therefore, cells were allowed to accumulate [U-14C]FB1 for 2 h and then PBS was removed and cultures were quickly rinsed 3 times with cold PBS and digested in 0.2 N NaOH. Samples of the digest were taken for both protein determination (Lowry et al., 1951Go) and for determination of radioactivity using liquid scintillation counting.

In order to determine if there was any metabolism of the accumulated FB1, confluent cultures (n = 4) were exposed to 1.35 mM FB1 containing a trace of [U-14C]FB1 for 4 h and then PBSG was removed and cells were rinsed 3 times in cold PBS, 200 µl of water added and the culture dishes were rapidly frozen at –80°C and then slowly thawed. Cells were detached with a disposable cell scraper, and the water/cell mixture collected in 1.5 ml polypropylene centrifuge tubes. The culture dishes were rinsed with 2 additional 200 µl volumes of water and the rinses combined (600 µl total). An equal volume of chloroform (600 µl) was added to each tube and the contents gently mixed and then centrifuged to achieve phase separation. The aqueous layer was removed and the chloroform layer washed with 200 µl of water. The combined water extracts were dried under vacuum. The residues were dissolved in 40 µl of water. Radioactivity in 10 µl of the water fraction was determined by liquid scintillation counting. The remaining chloroform extracts were taken to dryness and the radioactivity determined. Samples of the PBSG-[U-14C]fumonisin solutions from each culture dish (n = 3) were also evaporated to dryness under vacuum, dissolved in water and the radioactivity determined and then the volumes adjusted with water so as to have approximately the same amount of radioactivity/µl as the cell extracts. The actual FB1 content of the dosing solutions and the aqueous cell extracts was determined by high performance liquid chromatography (HPLC) using the method of Meredith et al. (1996). The FB1 in the cell extracts, determined by HPLC analysis, was compared to the calculated FB1 based on the specific activity in the solutions with which the cells were treated. Differences in the actual and calculated FB1 content would indicate FB1 metabolism.

The kinetics of efflux was measured by allowing cells to accumulate 50 µM [U-14C]FB1 for 4 h in DMEM/Ham's F12 and then replacing the medium with DMEM/Ham's F12 but without FB1. Cells were harvested and the protein content and radioactivity determined as described for the uptake experiment. Sample times for the [U-14C]FB1 efflux experiment were less than or equal to 60 min based on preliminary experiments (Riley et al., 1998Go).

The effect of FB1 on accumulation and persistence of free sphingoid bases in cells was determined by measuring the change in free sphinganine in confluent cultures. All experiments were conducted with DMEM/Ham's F12 plus 2% fetal calf serum. Stock solutions of FB1 were dissolved in growth medium (final concentration 25 µM FB1) and added to the confluent cultures on day 3 (72 h after seeding) and cells were allowed to accumulate free sphingoid bases for 48 h at which time the culture medium was removed and replaced with culture medium (DMEM/Ham's F12 plus 2% fetal calf serum) containing either no additions or 150 nM ISP-1. ISP-1 is an inhibiter of the first enzyme (serine palmitoyltransferase) in the de novo sphingolipid biosynthetic pathway (Riley and Plattner, 2000Go). Inhibition of this enzyme prior to addition of the ceramide synthase inhibitor, FB1, has been shown to prevent (or reduce in vivo) the accumulation of free sphingoid bases in both cultured renal epithelial cells and mouse kidney (Riley et al., 1999bGo). After removing the FB1, the cultures with and without ISP-1 were harvested over a 24 h period by placing the dishes on ice, rinsing twice with phosphate buffered saline (PBS) and then scraping the cells off the surface of the dishes using a rubber spatula. Cells were collected in 1.5 ml polypropylene tubes and pelleted by centrifuging at 4000 x g for 10 min at 4°C. The PBS was removed and the cells frozen at –20°C until sphingolipid analysis. Additional dishes of cells for each treatment were digested in 0.2 N NaOH and analyzed for total protein by the Lowry method (Lowry et al., 1951Go). In a separate experiment, conducted under identical conditions, LLC-PK1 cells were exposed to 25 µM FB1 continuously for 96 h to determine the time course for accumulation of free sphingoid bases.

FB1 and ISP-1.
FB1 (> 95% purity) was prepared as described by Meredith et al. (1996). The serine palmitoyltransferase inhibitor ISP-1 (> 99% pure), was prepared as described by Riley and Plattner (2000). Radiolabelled [U-14C]FB1 (specific activity 1.6 µCi/mg; >95% purity) was prepared using [1,2–14C]acetate as described by Blackwell et al. (1994). The C20-sphinganine internal standard used to quantitate free sphingoid bases in the sphingolipidanalyses described below, was prepared and generously provided by A. H. Merrill, Jr. and D. C. Liotta, Emory University, Atlanta, GA.

Sphingolipid analyses.
Free sphinganine and free sphingosine in base-treated lipid extracts of mouse tissues and LLC-PK1 cells were determined by HPLC as described in Riley et al. (1999a). Sphingoid base concentrations were normalized to the protein content using the bicinchoninic acid reagent (Pierce Inc., Rockford, IL) for mouse tissues and the Lowry method (Lowry et al., 1951Go) for LLC-PK1 cells. Total complex sphingolipids in mouse tissues were also determined using the procedures described in Riley et al. (1999a).

Statistical analysis.
Statistical analysis was done using Sigma Stat software (Jandel Scientific, San Rafael, CA). One-way ANOVA was used followed by Dunnet's or Newman-Keuls test for post hoc multiple comparison. All data were expressed as mean ± SD, and differences among means were considered significant if the probability was <=0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies in Vivo
Following a single po dose of 25 mg FB1/kg body wt (Fig. 2Go), there was a significant (p < 0.05) time-dependent increase in free sphinganine in the epithelial cells harvested from the small intestines that peaked at 4 to 12 h and declined to control levels by 48 h postdosing. Apart from the significant drop at 24 h free sphingosine levels were constant for the first 12 h and then dropped significantly at 24 to 48 h. The ratio of sphinganine to sphingosine was increased between 4 and 24 h and fell to the control level by 48 h. There were no statistically significant differences in total complex sphingolipids over the 48 h period (data not shown).



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FIG. 2. The time course (0–48 h) of the change in free sphingosine (A) and free sphinganine (B) and the free sphinganine to sphingosine ratio (insert B) in small intestine epithelial cells of mice given a single gavage dose of FB1 (25 mg/kg body weight). The time zero mice received saline. Free sphingosine and free sphinganine contents at various time points were determined by HPLC-fluorescence detection (Riley et al., 1999aGo). The values are expressed as the mean ± SD (n = 3 to 7/time point). Asterisks indicate means that are significantly different (p <= 0.05) from zero time point.

 
In liver (Fig. 3Go) there was a significant (p < 0.05) time-dependent increase in free sphinganine levels with a peak at 4 to 12 h and decline to control levels by 24 h. This paralleled the time-dependent changes in sphinganine seen in the epithelial cells of the small intestines (Fig. 2Go). The ratio of sphinganine to sphingosine was not altered due to significant increases in free sphingosine that paralleled the changes in free sphinganine. Total complex sphingolipid levels were unchanged over the 48 h period (data not shown).



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FIG. 3. The time course (0–48 h) of the change in free sphingosine (A) and free sphinganine (B) and the free sphinganine to sphingosine ratio (insert B) in liver. All other details as in Figure 2Go.

 
In the kidney (Fig. 4Go) free sphinganine levels and the ratio of sphinganine to sphingosine were significantly increased at 4 h and maximally increased at 8 h. Free sphinganine decreased slowly after 8 h but remained significantly elevated over the entire 48 h period. The increase in free sphingosine, paralleled the free sphinganine increase, however, returned to control levels at 48 h. The increase in free sphinganine in kidney was much greater than in either liver or the epithelial cells of the small intestines. In a follow-up experiment, it was found that the elevated free sphinganine and the ratio of sphinganine to sphingosine returned to control values (they were not significantly greater than the time zero values) after 120 h (Fig. 5Go). The free sphingosine was at the time zero value at 96 h but was slightly elevated at 120 h (Fig. 5AGo), but within the range seen historically in untreated mouse kidney. Liver and epithelial cells of the small intestines were also analyzed (data not shown) with results similar to that seen in Figures 2 and 3GoGo. Similar to the liver and epithelial cells of the small intestines, no changes in total complex sphingolipid levels were seen in kidney over the test period (data not shown).



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FIG. 4. The time course (0–48 h) of the change in free sphingosine (A) and free sphinganine (B) and the free sphinganine to sphingosine ratio (insert B) in kidney. All other details as in Figure 2Go.

 


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FIG. 5. The time course (0–120 h) of the change in free sphingosine (A) and free sphinganine (B) and the free sphinganine to sphingosine ratio (insert B) in kidney. All other details as in Figure 2Go.

 
In kidney, a single high dose of FB1 (25 mg/kg bw) followed by a low dose (0.03 mg/kg bw/day) prolonged the time (> 120 h) required for free sphinganine and the ratio of sphinganine to sphingosine to return to control levels (Fig. 6Go). The low dose alone did not cause an increase in free sphinganine, sphingosine, or the ratio of sphinganine to sphingosine after daily gavage for 5 days. The level of free sphinganine and the ratio of sphinganine to sphingosine were similar to that seen at 72 h in Figure 5Go. Assuming that the kinetics of the initial increase and subsequent decrease following the high dose were similar to previous experiments, then the 4 day exposure to the low level (0.03 mg/kg bw/day) of FB1 would prolong the return to control levels for an additional 48 h (predicted return to control levels at 168 h).



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FIG. 6. The changes in free sphingoid bases (free sphingosine [filled bars] and free sphinganine [open bars]) and the free sphinganine to free sphingosine ratio (insert) in kidney. Mice were given a daily gavage dose of FB1 (0.03 mg/kg body weight) or saline (0 mg/kg body weight/day) for 4 days following an initial single gavage dose of FB1 of either 25 mg/kg body weight or 0.03 mg/kg body weight or saline (0 mg/kg body weight). Animals were gavaged daily for 5 days and then killed 120 h after the first dose. The values are expressed as the mean ± SD (n = 6 to 8/ treatment). All other details as in Figure 2Go.

 
Studies in Vitro
At concentrations between 4 and 1000 µM [U-14C]FB1, uptake by cultured renal epithelial cells was linear following first order kinetic (Fig. 7AGo). The amount of the FB1 (based on the HPLC analysis) recovered from the aqueous extracts of cells allowed to accumulate FB1 for 4 h was the same as the calculated amount of accumulated FB1 based on the specific activity of [U-14C]FB1 in the solution with which the cells were treated (Table 1Go). [U-14C]FB1 levels in LLC-PK1 cells reached an apparent equilibrium with the extracellular [U-14C]FB1 concentration after 2- to 8-h exposure (Fig. 7BGo). The half-life for efflux of [U-14C]FB1 from renal epithelial cells after removal of FB1 from the culture medium was approximately 2 to 3 min (Fig. 7AGo inset).



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FIG. 7. Uptake and efflux of FB1 in LLC-PK1 cells. (A) The uptake of [U-14C]FB1 by LLC-PK1 cells at various FB1 concentrations. Individual values are shown in the uptake experiment along with the first order regression analysis (r2 = 0.99, n = 20). Inset is the efflux of [U-14C]FB1 from confluent cultures expressed as a percentage of the time zero controls for the efflux experiment. Values for efflux are the mean ± SD, n = 3. (B) The time course of [U-14C]FB1 uptake at 14, 23, and 44 µM FB1 expressed as a fraction of the calculated equilibrium value based on a cell volume of 12.2 µl/mg protein (Riley et al., 1985Go). See the Material and Methods section for additional details.

 

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TABLE 1 Fumonisin B1 Content of LLC-PK1 Cultures
 
In cultured renal epithelial cells, free sphinganine accumulated rapidly, reaching maximal elevation after 24 h and remained elevated as long as FB1 was present (Fig. 8Go inset). When FB1 was removed the free sphinganine remained elevated over the entire 24 h period after removal of FB1 (Fig. 8Go). However, in the presence of the serine palmitoyltransferase inhibitor, ISP-1, the free sphinganine returned to the control value by 12 h (Fig. 8Go). A similar response was seen with free sphingosine levels (data not shown). The approximate half-life for the decrease in intracellular free sphinganine in the presence of ISP-1 after FB1 removal is between 1 and 2 h (Fig. 8Go), whereas the half-life in the absence of serine palmitoyltransferase inhibition after removal of FB1 is approximately 72 h (Riley et al., 1998Go).



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FIG. 8. The persistence of free sphinganine in LLC-PK1 cells after 48 h exposure to FB1 (25 µM) and then replacement with FB1 free medium (filled circles) or medium without FB1 and containing 150 nM ISP-1 (open circles). Inset is the time course for the increase in free sphinganine from a separate experiment conducted under identical conditions but with the exception that cells remained exposed to FB over a 96 h period. Values are means ± SD of 3–6 replicate samples.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inhibition of ceramide synthase (as evidenced by the increase in the upstream intermediate sphinganine) occurs very quickly after mice are dosed either subcutaneously (Enongene et al., 2000Go) or orally with FB1. The increase in free sphinganine in kidney was most pronounced; however, the increase in free sphinganine in liver and epithelial cells of the small intestines, while of lesser magnitude, occurred 2 h sooner (2 h vs. 4 h). Nonetheless, all the tissues examined appeared to be affected at about the same time.

As in the previous study using sc exposure to a single dose of FB1 (Enongene et al., 2000Go), increased sphinganine and sphingosine were rapidly reversed in the liver and the epithelial cells of the small intestines. As seen in the previous study, the accumulation of free sphingoid bases in kidney is much more persistent than in either liver or the intestinal epithelial cells. This is in close agreement with studies in rats that have shown that the increase in free sphinganine in liver and kidney is reversible (Voss et al., 1998Go) but is more persistent in kidney (Garren et al., 2001Go) than in liver. One implication of this finding is that when investigating changes in free sphingoid bases in liver, a 24 h fasting period may mask any changes that are induced by short-term exposures.

The rapid decrease of free sphingoid bases in liver suggests that the liver is much better equipped to either eliminate or metabolize free sphingoid bases. Alternatively, the opposite may be true in kidney or the kidney may retain free sphinganine or accumulate it from serum and may be more resistant to the toxic effects of free sphingoid bases. For example, results of in vivo studies indicate that in Sprague Dawley rats, hepatoxicity is associated with free sphinganine levels > 12 nmole/g fresh weight (Voss et al., 1996Go) and in mouse liver, increased apoptosis has been associated with free sphinganine levels greater than 12 nmole/g fresh weight (Delongchamp and Young, 2001Go). In the rat, nephrotoxicity was associated with higher sphinganine levels (> 129 nmole/g fresh weight), however, renal free sphinganine increased to much higher concentrations in kidney at lower FB1 doses than did hepatic free sphinganine concentration; 15 ppm FB1 and 50 ppm FB1 for kidney and liver, respectively (Voss et al., 1996Go). This is similar to the free sphinganine levels (free sphinganine > 100 ± 12 < 127 ± 18 nmole/g fresh wt) associated with significantly increased nephropathy and the free sphinganine level (free sphinganine > 5.3 ± 1.2 < 15.1 ± 3.6 nmole/g fresh wt) associated with significantly increased hepatopathy in male BALBc mice (Sharma et al., 1997Go; Tsunoda et al., 1998Go).

This study supports and extends the finding by Wang et al. (1999) that maintaining rats on a low FB1 diet (1 ppm FB1 in AIN 76 diet), which by itself was insufficient to cause an increase in free sphinganine in urine, prolonged the elevation of free sphinganine in urine that had been induced by feeding a diet containing 10 ppm FB1. The feeding of 1 ppm FB1 diet for 40 days was, however, sufficient to cause a slight, but statistically significant, elevation in free sphinganine and free sphingosine in rat kidney (Wang et al., 1999Go), whereas free sphingoid bases in mouse kidney were not elevated after gavage of 0.03 mg/kg body weight (equivalent to 0.2 ppm FB1 diet calculated according to Lehman, 1954Go) for 5 days (Fig. 6Go). Thus, a po dose insufficient to elevate free sphinganine in mouse kidney can prolong the sphinganine elevation in kidney caused by a higher dose. The possible contribution of a low level of FB1 from the rodent chow plus the FB1 administered by gavage could have had an additive effect. Nonetheless, the daily oral dose plus whatever FB1 may have been present in the rodent chow (< 0.4 ppm) was insufficient by itself to elevate free sphinganine, sphingosine, or the ratio in kidney. While the mechanism by which low levels of FB can prolong the elevation of free sphinganine in urine and kidney is unknown, it is possible to speculate. Accumulation of free sphinganine will only occur when the rate of sphinganine formation exceeds its rate of removal. In this model, the rate of removal is a function of the rate of ceramide biosynthesis, sphinganine degradation, and sphinganine efflux from the cell. Assuming that serine palmitoyltransferase activity is much greater than either sphinganine kinase activity or the processes that regulate sphinganine efflux from the cell, then it is ceramide synthase activity that prevents sphinganine accumulation in cells that are not exposed to fumonisin. Thus, sphinganine will only accumulate when the level of ceramide synthase inhibition exceeds some threshold. Following a high dose of FB, a low dose could push the level of ceramide synthase inhibition above the threshold of inhibition necessary to allow sphinganine to accumulate.

Once elevated, the increase in free sphingoid bases is quickly reversed in liver, a fact that could reflect the rapid elimination of FB1 in vivo (Norred et al., 1993Go). However, elimination of FB1 from rat kidney was equally rapid (Norred et al., 1993Go) suggesting that it is the differences in metabolism or elimination of sphingoid bases that is responsible for the persistence in kidney. Once sphingoid bases are accumulated in cells, they do not easily diffuse out of cells, as evidenced by the fact that dead cells collected from the urine of FB1-treated rats contain levels of free sphingoid bases that are similar to those in kidneys undergoing apoptosis in the same animals (Riley et al., 1994Go). Thus, it appears that the time required to reverse the increase in free sphingoid bases in FB1-treated animals is not due only to the rapid efflux of FB1 but is also dependent on the kinetics of sphingoid base metabolism via the degradative pathway.

The results of the experiments using cultured renal epithelial cells support the hypothesis that it is the kinetics of sphingoid base metabolism and not FB1 elimination or metabolism that is responsible for the reduction in free sphingoid bases in tissues. The accumulation of FB1 by LLC-PK1 cells is a passive process (Fig. 7Go) and the fact that there was no change in the specific activity of [U-14C]FB1 accumulated by LLC-PK1 cells is evidence that FB1 (as has been shown in vivo) is not metabolized to any appreciable extent. The proportionality between accumulated FB1 and the micromolar concentration of the extracellular FB1 was calculated to be 0.061 pmoles FB1/mg protein/min. The average cell volume of LLC-PK1 cells is 12.2 µl/mg of protein (Riley et al., 1985Go). Thus, assuming there is minimal metabolism, the calculated minimum time required to reach intracellular equilibrium with the extracellular FB1 concentration is approximately 3 h. The time course studies indicate that the actual time to equilibrium is from 2 to 8 h (Fig. 7BGo). Clearly, the rate of efflux is much faster than the time required to attain equilibrium (minutes vs. hours). Thus, the kinetics of uptake and efflux of FB1 probably has little to do with the persistence and reversal of the elevation in free sphingoid bases in these cells. Conversely, the rate of sphingoid base metabolism plays a major role in the ability of LLC-PK1 cells to reverse the effects of FB1 on intracellular sphingoid base concentration. Inhibition of serine palmitoyltransferase activity (the first and rate limiting enzyme in de novo sphingolipid biosynthesis) results in a rapid and complete reduction in free sphingoid bases in the LLC-PK1 cells. Clearly, the balance between the rates of sphinganine biosynthesis and degradation are responsible for the persistence of free sphinganine in these cells. Similar phenomena in vivo could explain the persistence of free sphinganine in rodent kidney and possibly the rapid reversal in liver and intestinal epithelial cells.

The fact that the elevation in free sphingoid bases is rapidly reversed in the liver could limit the effectiveness of the elevation of free sphinganine in serum as a biomarker for FB exposure in humans. However, in a study in vervet monkeys, a single gavage dose of FB1 (10 mg/kg bw) caused an elevation in the ratio of sphinganine to sphingosine in serum that was sustained for several weeks (van der Westhuizen et al., 2001Go). Whether this will be the case in humans is unknown; however, studies that utilize the elevation in free sphinganine as a biomarker in humans (for example Qiu et al., 2001; van der Westhuizen et al., 1999Go) will need to take into account the possibility that rapid reversal would reduce the window for using the elevation in sphingoid bases as a biomarker for FB exposure as has been pointed out also by Turner et al. (1999).

In conclusion, liver and intestinal epithelial cells appear to handle the elevation in free sphinganine quite differently than kidney. Differences in the ability to metabolize free sphingoid bases may allow for the persistence of free sphingoid bases in rodent kidney and also allow accumulation of much higher levels in kidney than in liver. Conversely, the kidney appears to be more resistant to the toxic effects of disrupted sphingolipid metabolism, possibly explaining the apparent tolerance to the accumulation of high levels of free sphingoid bases.


    ACKNOWLEDGMENTS
 
The authors greatly appreciate the assistance and training provided by Ms. Jency Showker of the Toxicology and Mycotoxin Research Unit.


    NOTES
 
1 To whom all correspondence should be addressed. Fax: (706) 546-3116.E-mail: rriley{at}saa.ars.usda.gov. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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