* Toxicology and Mycotoxin Research Unit, R. B. Russell Research Center, USDA/ARS, P.O. Box 5677, Athens, Georgia 30604-5677;
College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602; and
Department of Chemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6
Received November 29, 2001; accepted February 1, 2002
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ABSTRACT |
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Key Words: fumonisin; fusarium verticillioides; sphingolipids; liver; kidney; gastrointestinal; ceramide synthase; serine palmitoyltransferase; ISP-1; LLC-PK1 cells.
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INTRODUCTION |
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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., 1993). A similar persistent retention of trace amounts of [14C]FB1 has been seen in cultured renal epithelial cells (Riley et al., 1998
). 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., 2001
). 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., 1998
). 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.
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MATERIALS AND METHODS |
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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., 2000), 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., 1992). 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., 1951) 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., 1998).
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, 2000). 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., 1999b
). 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., 1951
). 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 [1,214C]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., 1951) 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.
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RESULTS |
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DISCUSSION |
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As in the previous study using sc exposure to a single dose of FB1 (Enongene et al., 2000), 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., 1998
) but is more persistent in kidney (Garren et al., 2001
) 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., 1996) and in mouse liver, increased apoptosis has been associated with free sphinganine levels greater than 12 nmole/g fresh weight (Delongchamp and Young, 2001
). 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., 1996
). 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., 1997
; Tsunoda et al., 1998
).
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., 1999), 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, 1954
) for 5 days (Fig. 6
). 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., 1993). However, elimination of FB1 from rat kidney was equally rapid (Norred et al., 1993
) 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., 1994
). 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. 7) 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., 1985
). 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. 7B
). 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., 2001). 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., 1999
) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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