ToxSci Advance Access originally published online on September 7, 2006
Toxicological Sciences 2006 94(2):388-397; doi:10.1093/toxsci/kfl102
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Ceramide Synthase Inhibition by Fumonisin B1 Treatment Activates Sphingolipid-Metabolizing Systems in Mouse Liver
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602-7389
1 To whom correspondence should be addressed at: 10513 West 84th Terrace, Lenexa, KS 66214-1643. Fax: +1 (913) 685-8856. E-mail: qhe{at}cydexinc.com.
Received July 20, 2006; accepted August 29, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sphingolipids are important components of cell structure and cell signaling. Both external and internal stimuli can alter levels of cellular sphingolipids by regulating enzyme activities associated with sphingolipid metabolism. Fumonisin B1, mycotoxin produced by Fusarium verticillioides, is a reportedly specific inhibitor of ceramide synthase. In order to test our hypothesis whether ceramide synthase inhibition by fumonisin B1 alters other sphingolipid-metabolizing enzymes, we investigated the changes in free sphingoid bases and sphingomyelin (SM) and activities of key enzymes for their metabolism, sphingomyelinase (SMase), serine palmitoyltransferase (SPT), and sphingosine kinase (SPHK) in mouse liver. The hepatic free sphingoid bases increased significantly following five daily treatments with fumonisin B1 in mice. The activity of acidic SMase was enhanced by fumonisin B1, accompanied with a decrease in liver SM content. The expression and activities of SPT and SPHK1 in liver increased significantly following fumonisin B1 treatment. Another hepatotoxicant acetaminophen caused liver regeneration similar to fumonisin B1 but did not produce similar effects on liver sphingolipid-metabolizing enymes, suggesting that activation of sphingolipid metabolism was not a consequence of hepatocye regeneration. Data suggest that ceramide synthase inhibition by fumonisin B1 treatment stimulates sphingolipid-metabolizing systems to maintain a balance of cellular sphingolipids.
Key Words: sphingomyelinase; fumonisin; sphingolipid; sphingosine kinase; serine palmitoyltransferase.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sphingolipids are ubiquitous components of eukaryotic cell membrane structures, providing integrity to cellular membranes. There is increasing evidence for the involvement of sphingolipids in regulating various cellular functions, such as cell-cell interactions, cellular protein and receptor functions, membrane transport, and signal transduction (Prieschl and Baumruker, 2000
). Simple sphingolipid compounds, such as sphingosine and sphingosine-1-phosphate, have been shown to mediate cellular signal cascades of apoptosis and proliferation (Spiegel and Milstien, 2002).
Since both complex sphingolipids and the free sphingoid bases have important structural and functional properties in the cell and fumonisin B1 is reportedly a potent inhibitor of ceramide synthase (see below) causing a rapid accumulation of sphingosine (Wang et al., 1991), the resultant effect on sphingolipid homeostasis after fumonisin B1 treatment are worthy of investigation. Effect of fumonisin B1 on sphingolipid-metabolizing enzymes other than ceramide synthase has not been adequately studied. As illustrated in Figure 1, the sphingolipid synthesis starts with the condensation of L-serine and palmitoyl coenzyme A (palmitoyl-CoA) to 3-ketosphinganine, and its reduction to sphinganine in the endoplasmic reticulum. Serine palmitoyltransferase (SPT), a membrane-associated heterodimer consisting of two gene products, long-chain base (LCB) 1 and LCB2, is the rate-limiting enzyme for the sphingolipid synthesis (Hanada, 2003). Ceramide is central molecule that serves as the precursor for all major sphingolipids, that is, sphingomyelin (SM), glucosylceramide, and more complex sphingolipids in eukaryotic cells, and sphingolipid metabolism involves different key enzymes (Hannun et al., 2001).
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Fumonisin B1, a mycotoxin produced by Fusarium verticillioides, is reportedly a specific and potent inhibitor of ceramide synthase. The concentration of fumonisin B1 to inhibit 50% of activity of ceramide synthase (IC50) was estimated to be 0.1µM for rat liver microsomes (Wang et al., 1991
); and the IC50 was about 0.7µM for labeling of SM in neurons (Merrill et al., 1993). Inhibition of ceramide synthesis following fumonisin B1 exposure results in decrease of SM, other complex sphingolipids, and diacylglycerol (Wu et al., 1995; Yoo et al., 1996), while free sphingoid bases and their 1-phosphates are increased (Merrill et al., 2001). In yeast, fumonisin B1 inhibited ceramide synthase without any effects on inositol phosphorylceramide synthase, phosphatidylinositol synthase, and choline kinase, though fumonisin B1 decreased levels of these lipids (Wu et al., 1995). Long-term incubation of NIH/3T3 fibroblasts with fumonisin B1 inhibited ceramide synthase leading to decrease of sphingolipids and glycosphingolipid but increased the activities of three glycosyltransferases in the pathway of globotriaosylceramide (Gb3) synthesis, while the activities of ganglioside 3 (GM3) and SM synthase were not affected (Meivar-Levy et al., 1999). We have recently reported that activity of hepatic SPT was increased in response to fumonisin B1 treatment in mouse liver (He et al., 2004). It is unknown whether other enzymes for sphingolipid metabolism are affected following fumonisin B1 exposure.
Following fumonisin B1 treatment, the cells undergo growth arrest and apoptosis (Jones et al., 2001; Sharma et al., 1997; Yoo et al., 1996). Compensatory cellular proliferation was observed in liver and kidney after exposure of rodents to fumonisin B1 (He et al., 2004; Howard et al., 2001b, Sharma et al., 2003). In some cases, fumonisin B1 is mitogenic and antiapoptotic (Schroeder et al., 1994). Due to its reportedly specific inhibition of ceramide biosynthesis, fumonisin B1 has been used extensively to study the biological roles of ceramide and complex sphingolipids in different cell cultures (Le Stunff et al., 2002; Reiss et al., 2004).
In the present study, we hypothesized that ceramide synthase inhibition by fumonisin B1 will influence the enzymes involved in sphingoid base metabolism in order to reestablish the homeostasis of lipid messengers. Therefore, the activities of sphingomyelinase (SMase), SPT, and sphingosine kinase (SPHK) in mouse liver were analyzed after fumonisin B1 treatment. We observed that the activities and expression of both hepatic SPT and SPHK were increased in response to fumonisin B1 treatment. Fumonisin B1 treatment resulted in enhanced activity of acidic but not neutral SMase (N-SMase). Results suggest that ceramide synthase inhibition by fumonisin B1 is associated with perturbation of sphingolipid metabolism by other metabolizing enzymes, and in most cases, maximal effects on sphingolipid-metabolizing enzymes were observed at the relatively low dose of fumonisin B1.
Since induction of cellular enzymes may be related to liver regeneration, we compared the effect of another hepatotoxicant, acetaminophen, on the synthesis of sphiongolipid-metabolizing enzymes. Although acetaminophen caused liver regeneration to an extent comparable to that with fumonisin B1, the former had no influence on the synthesis of SPT subunits or SPHK1.
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MATERIALS AND METHODS |
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Materials.
Fumonisin B1 (> 98% purity) was obtained from Program on Mycotoxins and Experimental Carcinogenesis (Tygerberg, South Africa). C20-sphinganine standard (D-erythro-C20-dihydro-sphingosine, 98% purity) was procured from Matreya Inc., (Pleasant Gap, PA). Polyclonal SPT antibody against an amino acid sequence corresponding to 121–238 residues of LCB1 (mouse origin) was from BD Biosciences (Rockville, MD). Polyclonal antibody against SPHK1 was purchased from Oncogen (San Diego, CA). All other reagents were purchased from Sigma-Aldrich Chemical Company Inc., (St Louis, MO), unless otherwise stated.
Animal treatments and sampling.
Female mice were employed, as they are more sensitive to hepatotoxic effects of fumonisin B1 than male counterparts (Howard et al., 2001a). Six-weeks-old female C57BL/6N mice weighing about 22 g were procured from Harlan Laboratories (Indianapolis, IN). They were acclimatized for 1 week before experiments under controlled environmental conditions of 23°C and 65% relative humidity, with a 12-h light/dark cycle. Feed and water were provided ad libitum. Animals were treated with humane care following the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee.
Mice were divided randomly into four groups with five animals each. The mice were treated with daily sc injection of 0, 0.75, 2.25, or 6.75 mg/kg body weight fumonisin B1 for 5 consecutive days. The doses of fumonisin B1 were based on our previous study where a dose-dependent hepatopathy was observed (Tsunoda et al., 1998). Sterilized phosphate buffered saline (PBS) was used as the solvent for fumonisin B1 and for injecting control groups. During treatment, body weight, food, and water consumptions were recorded daily.
Twenty-four hours following the final fumonisin B1 treatment, mice were sacrificed under halothane anesthesia. Livers were collected from each animal and weighed; aliquots of randomized liver tissue were frozen in liquid nitrogen and stored at – 85°C until analysis.
Analysis of hepatic damage.
The liver injury following fumonisin B1 treatment was determined by estimation of plasma aniline aminotransferase (ALT) and aspartate aminotransferase (AST) activities and numeration of apoptotic hepatocytes in liver tissue. The activity of plasma ALT and AST was analyzed using a Hitachi 912 Automatic Analyzer (Roche Diagnostics, Indianapolis, IN). Liver tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned (4–5 µm) for terminal dUTP nick end labeling (TUNEL) with a peroxidase-based In Situ Cell Death Detection kit (Roche Diagnostics) as described previously (Sharma et al., 1997).
Sphingolipid analysis.
Free sphingosine and sphinganine of liver in base-treated lipid extracts were determined by high-performance liquid chromatography (HPLC) using a modification of the extraction methods described previously (Merrill et al., 1988). Sphingoid bases were quantified based on the recovery of a C20-sphinganine standard (D-erythro-C20-dihydro-sphingosine). The HPLC apparatus and derivation procedure were similar to those indicated previously (He et al., 2004).
SM and SMase assay.
SM was analyzed by a fluorescence-based method following enzymatic catalysis (He et al., 2002). Livers were homogenized in 0.25% Triton X-100 in PBS (1:20) and centrifuged (10,000 x g for 5 min). Aliquots of 20 µl supernatant were mixed with equal volumes of homogenizing buffer followed by heating at 70°C for 5 min to destroy endogenous enzymatic activity. Ten microliters of the supernatant was added to an enzyme cocktail consisting of 12.5 mU of Bacillus cereus SMase, 400 mU of alkaline phosphatase, 120 mU choline oxidase, 200 mU of horseradish peroxidase, and 20 nmol of 10-acetyl-3-dihydrophenoxazine, a sensitive fluorogenic probe for hydrogen peroxide (Amplex red reagent, Molecular Probes, Inc., Eugene, OR), in a reaction buffer (0.1M Tris-HCl, 10mM MgCl2, pH 7.4). For each sample, a negative control containing 10 µl of the sample and the same reaction mixture without SMase was employed. After 20 min incubation at 37°C, the microplate was read using a fluorescence reader (Molecular Devices Corp., Sunnyvale, CA), with the excitation and emission wavelengths set at 560 and 590 nm, respectively. SM contents were calculated from the difference in fluorescence between the test and the negative control samples by comparing with a standard curve.
The activity of SMase was determined using Amplex Red Sphingomyelinase Assay Kit according to the manufacturer's protocol (Molecular Probes). Frozen tissue was homogenized in a buffer containing 100mM Tris-HCl, 0.5% Triton X-100 (pH 7.4). The homogenate was then centrifuged at 10,000 x g for 5 min, and the supernatant (50 µg protein) was used for SMase assay. The N-SMase activity was determined in 100 µl of reaction solutions containing 0.1M Tris-HCl (pH 7.4), 10mM MgCl2, 0.5mM SM, 0.2 U/ml choline oxidase, 2 U/ml horseradish peroxidase, 8 U/ml alkaline phosphatase, and 0.2mM Amplex Red. For acidic SMase (A-SMase), the reaction was carried out in an acidic buffer of 50mM sodium acetate (pH 5.0) containing 0.5mM SM without Mg2+, and then the reaction was detected with Amplex reagents mixed in 100mM Tris-HCl (pH 8.0, to bring up pH to the optimal point for Amplex reagents). The excitation and emission wavelengths were 560 and 590 nm, respectively. The reaction was linear in a range of up to 160 µg protein for 30 min incubation of the sample with reaction buffer at 37°C. In all experimental reactions for SMase assay, the samples were allowed to incubate with reaction buffer for 20 min at 37°C. SMase contents were calculated based on a standard curve prepared parallel to sample reactions. The standard curve was in linear range up to 10 mU/ml of SMase in the reaction buffer.
The protein contents were determined by Bradford reagent according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin (BSA) as a standard.
Expression of genes for SPT and SPHK.
Gene expression of SPT subunits (LCB1 and LCB2) and SPHK isoforms (SPHK1 and SPHK2) in liver was analyzed by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR). The frozen tissue was homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH). The first-strand cDNA was synthesized using SuperscriptIII reverse transcriptase and Oligo(dT)12–18 primer (InVitrogen Life Technologies, Carsbad, CA). The abundance of mRNA for selected genes in liver tissues was analyzed by PCR using Taq DNA polymerase and 0.2µM of each primer in 1x PCR buffer containing 2mM MgCl2. The PCR reactions were performed in an Eppendorf Mastercycler Gradient (Eppendorf Scientific Inc., Westbury, NY). Table 1 shows the sequences of primers (chosen by Primer3 program, Whithead Institute, Cambridge, MA) for respective genes and conditions employed for amplification reaction. The PCR products were separated on 2% agarose gel containing ethidium bromide and detected by an ultraviolet transilluminator (Ultra Lum Inc., Carson, CA). Images were captured using a Kodak DC290 camera followed by digitization using UN-SCAN-IT software (Silk Scientific Inc., Orem, UT). Density of bands for ß-actin in the same sample was used to normalize the expression of each gene. The quantitative validity of RT-PCR has been confirmed by either northern blot in lipopolysaccharide (LPS)-treated J774.A macrophages (He et al., 2001) or ribonuclease protection assay in mouse liver (Bhandari and Sharma, 2002).
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Protein levels of SPT and SPHK.
Frozen liver tissues were homogenized in lysis buffer (20mM Tris-HCl pH 8.0, 137mM NaCl, 2mM ethylenediaminetatraacetate [EDTA], 10% glycerol, 1% Triton X-100, 100µM dithiothreitol, 1mM sodium orthovanadate, 20µM phenylmethylsulfonyl fluoride, and 10µg/ml of each leupeptin and aprotinin). Lysates were centrifuged for 30 min at 42,000 x g, and supernatants were collected for Western blot analysis after quantifying protein concentrations. An aliquot of 10 µg protein from each sample was subject to electrophoresis on polyacrylamide gels containing 10% sodium dodecylsulfate and then transferred to polyvinylidene difluoride membranes. Equal loading and transfer of protein was confirmed by staining the membranes with reversible Ponceau S before incubation with respective primary antibodies. Proteins were detected with polyclonal antibodies, against LCB1, LCB2, and SPHK1, respectively, using enhanced chemiluminescence detection kit (Amersham Pharmacia, Piscataway, NJ). The exposure time of the film was optimized to be in the linear range before the saturation occurs.
Activity of SPT.
The activity of SPT in liver was analyzed using the method described earlier (Williams et al., 1984) with minor modification. Briefly, the frozen tissues were homogenized in homogenization buffer (50mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 5mM DL-dithiothreitol, 10mM EDTA, and 0.25M sucrose, pH 7.4), and homogenates were centrifuged at 30,000 x g for 30 min. Aliquots of 50 µg protein in the supernatant were used for analysis of SPT activity, as described (Williams et al., 1984) using [3H]-serine (Moravek, Brea, CA) and palmitoyl-CoA (Sigma) as substrates and expressed as pmol/min/mg protein.
Activity of SPHK.
The activity of SPHK in liver homogenate was estimated according to the method described previously (Olivera et al., 2000). The protein extract (50 µg protein for each reaction) from liver homogenate was incubated in reaction buffer (20mM Tris, pH 7.4, 20% glycerol, 1mM mercaptoethanol, 1mM EDTA, 1mM sodium orthovanadate, 40mM ß-glycerophosphate, 15mM sodium fluoride, 1mM phenylmethylsulfonyl fluoride, and 10 µg/ml of each leupeptin and aprotinin) containing 50µM sphingosine as a sphingosine-BSA complex in a total volume of 190 µl. The reaction was started by adding 10 µCi of 
-32P-ATP (10 µl, 20mM, Amersham) containing MgCl2 (200mM) and incubated for 15 min at 37°C. The labeled lipids in the organic phase were resolved by thin-layer chromatography on Silica Gel G60 (EMD Chemical, Gibbstown, NJ). Conditions to ensure linearity of the enzymatic reaction with time of incubation and protein concentration were optimized in preliminary trials.
Gene expression of SPT and SPHK in liver regeneration after acetaminophen treatment.
Since the response to fumonisin B1 in mice includes cell damage and hepatic regeneration (He et al., 2004; Sharma et al., 2003), the expression of SPT and SPHK during regeneration was determined after treatment of female mice with acetaminophen (a well-known hepatotoxicant) injections. In a separate experiment, groups of five female mice each, similar to ones as above, were injected with 2.25 mg/kg fumonisin B1 sc or with 200 mg/kg of acetaminophen (10 mg/ml at 40°C) in PBS ip once daily for 5 days. Concurrent control groups for both fumonisin B1 and acetaminophen were injected with PBS using the route similar to the respective treatments. Animals were sampled one day after, and hepatocyte proliferation was evaluated in liver sections by expression of proliferating cell nuclear antigen (PCNA) as described earlier (Sharma et al., 2003). The expression of LCB1, LCB2, and SPHK1 in liver was determined by RT-PCR as indicated above.
Statistical analysis.
Results are presented as mean ± SE. Data were analyzed by one-way ANOVA followed by Duncan multiple range tests unless otherwise stated in the text. All statistical analyses were done using the SAS program (SAS, Cary, NC). The level of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with Fumonisin B1 Caused Liver Damage
Treatment of the animals with fumonisin B1 for 5 days did not cause changes in body weights and liver weights and in the ratio of liver over body weight (data not shown). The treatment resulted in 4- to 20-fold increases in activities of plasma ALT and AST compared to the saline-treated controls (Table 2), indicating liver injury. The hepatic damage was also confirmed by the increased number of apoptotic hepatocytes detected with TUNEL staining on liver sections (Table 2). For most parameters, however, there seemed to be a threshold at 0.75 mg/kg dose after which there was no remarkable increase in the effects.
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Accumulation of Free Sphingoid Bases in Liver Following Fumonisin B1 Treatment
It is known that ceramide synthase inhibition following fumonisin B1 exposure results in accumulation of free sphingoid bases. After five daily treatments of mice with fumonisin B1, the dose-dependent increases of hepatic free sphingosine and sphinganine leveled of at the middle dose of 2.25 mg/kg (Table 3).
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Alteration of SM and SMase in Liver
SM can be metabolized to ceramide by SMase. It is known that ceramide synthase inhibition by fumonisin B1 reduced both ceramide and SM in cells (Merrill et al., 2001
). To see whether SM and SMase could be affected following a short-term exposure of mice to fumonisin B1 in vivo, the content of SM and activity of SMase were analyzed. Results showed that hepatic SM was reduced about 60% compared to controls in response to fumonisin B1, with maximum decrease again at the lowest dose. A dose-dependent increase in activities of A-SMase was observed with a significant increase at the highest dose of fumonisin B1 (Fig. 2). The changes of N-SMase in response to fumonisin B1 were not significant.
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Increase of Hepatic SPT and SPHK Gene Expression Following Fumonisin B1 Treatment
We have reported that fumonisin B1 treatment increased activity of liver SPT (He et al., 2004
). To explore the possible mechanisms for the induction of SPT following fumonisin B1–induced ceramide synthase inhibition, we analyzed the expression of SPT in liver. The gene expression of LCB1 and LCB2, two subunits of SPT, was significantly increased (Fig. 3). It has been reported that fumonisin B1 increased 1-phosphate metabolites of free sphingoid bases (Merrill et al., 2001). The increase in sphingoid base-1-phosphate could result from increased input of free sphingoid bases per se, or activation of SPHK, or both. It was therefore of interest to determine if fumonisin B1 treatment could alter SPHK expression. Results showed that gene expression of SPHK1 increased significantly in response to fumonisin B1 treatments, with the increase reaching its maximal at the lowest dose of 0.75 mg/kg employed here (Fig. 4). In contrast, expression of SPHK2 was not affected by fumonisin B1 treatment.
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Levels of SPT Subunits and SPHK1
Levels of SPT subunits and SPHK1 determined by Western blots are depicted in Figures 5 and 6. Since gene expression of SPHK2 were unaltered after fumonisin B1 treatment, the protein levels for this isoform were not analyzed. Data shown in Figures 5 and 6 indicate that there was a lack of relationship between the dose of fumonisin B1 used and the protein levels of the induced enzymes, which were maximally stimulated at the lowest dose of fumonisin B1 tested (0.75 mg/kg per day), as was also observed with gene expression.
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Activities of SPT and SPHK Increased with the Protein Expression
Consistent with increases in protein expression, the activities of both SPT and SPHK were increased after fumonisin B1 treatment in mouse liver (Fig. 7). Again, there was a lack of dose-response relationship for both enzyme activities. Similar to the increases of protein levels, the activities of both enzymes reached their highest levels at 0.75 mg/kg; however, the increases were about fourfold and twofold of controls for SPT and SPHK, respectively.
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Induction of SPT and SPHK Was Not Due to Hepatic Regeneration
Mice injected with acetaminophen showed proliferation of PCNA-positive hepatocytes similar to that observed with fumonisin B1 and also described earlier (He et al., 2004
; Sharma et al., 2003); however, no effect of acetaminophen treatment was observed on the expression of LCB1, LCB2, or SPHK1 (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current study demonstrated that treatment of mice with fumonisin B1, a reportedly specific inhibitor of ceramide synthase, enhanced activities of liver SMase, SPT, and SPHK leading to compensatory changes in sphingolipids and/or further perturbation of sphingolipid metabolism. Fumonisin B1 caused inhibition of sphingolipid biosynthesis, leading to accumulation of free sphingoid bases and decreases in ceramide, SM, and complex sphingolipids, as reported in a variety of previous studies (Merrill et al., 1993
; Wang et al., 1991; Yoo et al., 1996). The important novel findings in the current study are that expression and activities of SPT and SPHK were upregulated following fumonisin B1 treatment. This upregulation apparently was not a consequence of increased hepatic regeneration in response to fumonisin B1 toxicity, as treatment with a known hepatotoxicant, acetaminophen, induced liver regeneration demonstrated by an increase in PCNA in liver but did not induce the same effects on SPT and SPHK. Hepatic regeneration was accompanied by an increase of SM synthase activity during the first 18 h with a subsequent decrease of SM synthase and increase of SMase at 24 h following hepatectomy (Albi et al., 1999). It is unknown whether liver regeneration in partial hepatectomy affects SPT or SPHK. However, the cellular mechanisms of tissue regeneration under different conditions may be different, thus, contributing to the observed disparities.
The exact mechanisms of observed effects in this study are yet to be investigated. Enzymes that regulate bioactive lipids function as switches by regulating the levels of bioactive substrates and products (Hannun et al., 2001) and, therefore, regulate cellular signal transduction. Ceramide can be generated through SM hydrolysis by SMase. Ceramide is essential to cells and a decrease in intracellular ceramide as a result of de novo biosynthesis inhibition by fumonisin B1 could increase the activity of SMase to compensate for reduced cellular ceramide level by production of ceramide via SM hydrolysis. In mice treated with similar doses of fumonisin B1, no decrease of ceramide was noticed (Tsunoda et al., 1998), suggesting that stimulation of A-SMase activity compensated the generation of ceramide following ceramide synthase inhibition. In cell signaling involving ceramide, the A-SMase appears to be more important; L929 fibrosarcoma cells deficient in A-SMase were resistant to ceramide-mediated caspase-independent apoptosis (Thon et al., 2005).
A noteworthy observation here is that the effects of fumonisin B1 treatment on SPT and SPHK were maximized at the lowest dose, whereas the accumulation of sphinganine or sphingosine in liver was dose related only up to 2.25 mg/kg of fumonisin B1. At the highest dose of 6.75 mg/kg fumonisin B1, no further increase in sphinganine or sphingosine accumulation was observed. Previous studies have shown that the IC50 of fumonisin B1 for inhibition of labeling sphingosine in hepatocytes or SM in neurons were 0.1µM and 0.7µM, respectively (Merrill et al., 1993; Wang et al., 1991). Treatment of hepatocytes with 1µM fumonisin B1 reached an almost complete inhibition of ceramide synthase (Wang et al., 1991). Based on homogenous distribution in the tissues, the sc injection of fumonisin B1 at 0.75 mg/kg daily for 5 days could reach approximately the concentration for complete inhibition of ceramide synthase. Over 1 µg fumonisin B1/g tissue (
1.4 µmol fumonisin B1/kg tissue) was detected in liver of pigs 72 h after iv injection of 0.4 mg fumonisin B1/kg body weight (Prelusky et al., 1994). Whereas the maximal effect on SM concentration was observed at 0.75 mg/kg of fumonisin B1, the induction of SPT and SPHK reached a plateau at the lowest dose employed here. It may be inferred that either the maximal induction of SPT and SPHK required only a small increase in the levels of free sphingoid bases, or alternatively, the increase in these enzymes is independent of the accumulation of sphinganine and/or sphingosine. Other factors such as de novo biosynthesis inhibition of ceramide and/or gangliosides, and defective cellular responses resulting from cytotoxicity, as demonstrated in the current study that plasma activities of ALT and AST reached maximum at the lowest dose (0.75 mg/kg/day), may be responsible for the lack of a dose-response effect on these sphingolipid-metabolizing enzymes.
In the current study, the activity of SPT paralleled the increased expression of respective protein, suggesting that the induction of SPT is due to increased enzyme synthesis. In response to LPS and cytokines, such as tumor necrosis factor (TNF)-
and interleukin (IL)-1, the production and activity of SPT was increased with subsequent increase of sphingolipid biosynthesis in liver of Syrian hamster and HepG2 cells (Memon et al., 1998). In our previous studies, we have consistently observed that fumonisin B1 increased the expression of many cytokines including TNF- (Bhandari and Sharma, 2002; He et al., 2001). The increase in SPT production following fumonisin B1 treatment may be related to the induction of TNF- and other cytokines.
SPHK catalyzes phosphorylation of sphingosine to form sphingosine-1-phosphate, a key cell signal modulator (Spiegel and Milstien, 2002). It is known that fumonisin B1 treatment significantly increased formation of sphinganine-1-phosphate (Merrill et al., 2001). Recently, we reported that SPHK was an important factor in resistance to fumonisin B1 cytotoxicity in HEK 293 cells, a human embryonic cell line (Sharma et al., 2004). Since fumonisin B1 produces cellular apoptosis and proliferation (He et al., 2004; Howard et al., 2001b; Jones et al., 2001; Sharma et al., 1997), the activation of SPHK and subsequent formation of sphingoid base-1-phosphate following fumonisin B1 treatment could contribute to the cell proliferation needed for liver regeneration. In addition to inhibition of ceramide synthase and subsequent blockade of ceramide formation, generation of sphingoid base-1-phosphate as a result of SPHK induction might partly involve the protective effect of fumonisin B1 in stress-induced apoptosis (Reiss et al., 2004).
SPHK can be activated by various external stimuli including growth factors, TNF-, IL-1ß, and many others (Spiegel and Milstien, 2002). It has been reported that TNF- could interact with TNF-receptor–associated factor 2 to activate SPHK with subsequent production of sphingosine-1-phosphate (Xia et al., 2002) and thus protect cells from apoptotic death (Osawa et al., 2001; Xia et al., 1999). Exposure of animals to fumonisin B1 increases free sphingoid bases as well as TNF- production (He et al., 2001; Riley et al., 2001; Sharma et al., 2003). It is plausible that either sphingoid bases or TNF- stimulates expression of SPHK since TNF- is known to activate SPHK and NF
B, an important transcriptional factor.
The observed changes in hepatic expression of SPT and SPHK following fumonisin B1 administration may be relevant in cellular signaling and function. While the accumulated free sphingoid bases are generally apoptotic, their respective phosphates impart cell survival properties (Merrill et al., 2001; Spiegel and Milstien, 2002). Activation of SPHK leading to sphingoid base-1-phosphate production may partially counteract the cell death signals by fumonisin B1–induced sphingoid bases accumulation and may be necessary for tissue repair. The observations here therefore emphasize the complexity of lipid signaling and paradoxical effects produced by fumonisin B1 or resulting alterations in sphingolipid metabolism.
In summary, fumonisin B1 treatment increased activities of SPT and SPHK in mouse liver; the increases in activities of SPT and SPHK could be explained by upregulation of their transcriptional and translational levels. The events observed here may be related in part to the reported effects of fumonisin B1 on apoptosis, cell cycling (Johnson et al., 2003), or tissue regeneration. Current study indicates that interference with one step of sphingolipid metabolism (ceramide synthase inhibition) could affect other metabolizing enzymes leading to further regulation of sphingolipid homeostasis.
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ACKNOWLEDGMENTS |
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This work was supported in part by U.S. Public Health Service grant ES09403 from the National Institute of Environmental Health Sciences.
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