Accumulation Of Sphingoid Bases Research Articles

N-Acetylcysteine Alleviates Impaired Muscular Function Resulting from Sphingosine Phosphate Lyase Functional Deficiency-Induced Sphingoid Base and Ceramide Accumulation in Caenorhabditis elegans.

Sphingosine-1-phosphate lyase (SPL) resides at the endpoint of the sphingolipid metabolic pathway, catalyzing the irreversible breakdown of sphingosine-1-phosphate. Depletion of SPL precipitates compromised muscle morphology and function; nevertheless, the precise mechanistic underpinnings remain elusive. Here, we elucidate a model of SPL functional deficiency in Caenorhabditis elegans using spl-1 RNA interference. Within these SPL-deficient nematodes, we observed diminished motility and perturbed muscle fiber organization, correlated with the accumulation of sphingoid bases, their phosphorylated forms, and ceramides (collectively referred to as the "sphingolipid rheostat"). The disturbance in mitochondrial morphology was also notable, as SPL functional loss resulted in heightened levels of reactive oxygen species. Remarkably, the administration of the antioxidant N-acetylcysteine (NAC) ameliorates locomotor impairment and rectifies muscle fiber disarray, underscoring its therapeutic promise for ceramide-accumulation-related muscle disorders. Our findings emphasize the pivotal role of SPL in preserving muscle integrity and advocate for exploring antioxidant interventions, such as NAC supplementation, as prospective therapeutic strategies for addressing muscle function decline associated with sphingolipid/ceramide metabolism disruption.

An update on genotoxic and epigenetic studies of fumonisin B1

Fumonisins (FBs), a widespread group of mycotoxins produced byFusarium spp., are natural contaminants in cereals and foodstuffs. Fumonisin B1 (FB1) is the most toxic and prevalent mycotoxin of this group, and it has been reported that FB1 accounts for 70-80% of FBs produced by the mycotoxigenic strains. The mode of action of FB1 depends on the structural similarity with sphinganine/sphingosine N-acyltransferase. This fact causes an accumulation of sphingoid bases and blocks the sphingolipid biosynthesis or the function of sphingolipids. Diverse toxic effects and diseases such as hepatocarcinogenicity, hepatotoxicity, nephrotoxicity, and cytotoxicity have been reported, and diseases like leukoencephalomalacia in horses and pulmonary oedema in horses and swine have been described. In humans, FBs have been associated with oesophageal cancer, liver cancer, neural tube defects, and infantile growth delay. However, despite the International Agency for Research on Cancer designated FB1 as a possibly carcinogenic to humans, its genotoxicity and epigenetic properties have not been clearly elucidated. This review aims to summarise the progress in research about the genotoxic and epigenetics effects of FB1.

Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana

Beauveria bassiana is a critical entomopathogenic fungus for pest biocontrol, whose efficiency depends on fungal development and stress resistance. Unlike its revealed location in plasma membrane patches in other organisms, B. bassiana Sur7 specifically localized in vacuoles. This vacuolar Sur7 was previously demonstrated to affect stress tolerance, hyphal development and virulence. There, however, remain more mechanistic details to be explored. In this study, transcriptomics and metabolomics were applied to investigate the mechanism of vacuolar Sur7. Analyses of transcriptomics and metabolomics displayed many differentially expressed genes and abundant metabolites in response to Sur7 loss, respectively. Together with genes associated with vacuolar biofunction (including transportation and hydrolysis), the altered metabolites contributed to cell wall construction and stress resistance. Particularly, an N-acetylglucosamine-associated Brg1/Nrg1 pathway was enriched and partially affected by Sur7. Absence of Sur7 changed the expression level of Brg1/Nrg1 pathway-related transcript factors, which interfered with downstream phenotype of sporulation. In addition, Sur7 was involved in the accumulation of sphingoid bases, which may affect sphingolipid-related signaling pathway. Although experimental evidence is further required, our studies provide a preliminary framework for future exploring the regulatory mechanism of Sur7, and give a new version of metabolic agency connecting Sur7 and downstream signaling pathway.

Fumonisin B1 regulates LDL receptor and ABCA1 expression in an LXR dependent mechanism in liver (HepG2) cells

The metabolic toxicity of Fumonisin B1 (FB1) converges at the accumulation of sphingoid bases and reduced ceramide levels. Several studies have alluded to a hypercholesterolemic endpoint after FB1 exposure, yet the molecular mechanisms remain elusive. Cell surface receptors are important regulators of cholesterol metabolism by regulating influx of lipids and efflux of cholesterol. Western blot analysis showed that FB1 elevates the expression of ABCA1 (a cholesterol efflux promoter) in an LXR dependent mechanism. We further highlight the potential role of PCSK9 in the degradation of LDL receptor. These data provide important evidence for the mechanism underlying hypercholesterolemia in FB1 treated models. The disruption of lipid homeostasis by FB1 is beginning to shift away from canonical ceramide synthase inhibition, and this changed perspective may shed light on diseases caused by dysregulated cholesterol metabolism such as cancer initiation and promotion.

Endoplasmic reticulum stress-mediated autophagy activation attenuates fumonisin B1 induced hepatotoxicity in vitro and in vivo

Although pathological characteristics of fumonisin B1 are known to induce hepatic injury over prolonged periods, the cellular defense mechanisms against the detrimental effects of FB1 are still unknown. The underlying mechanisms of FB1 toxicity are thought to be related with the inhibition of ceramide synthase, causing an accumulation of sphingoid bases, which in turn cause development of oxidative stress. Herein, we investigated whether autophagy, a cellular defense mechanism, protects liver cells from FB1 exposure. To accomplish this, we utilized HepG2 cells and a mouse model to study the effects of FB1 in the autophagy pathway. FB1 was capable of inducing autophagy via the generation of ROS, induction of endoplasmic reticulum stress, phosphorylation of JNK, suppression of mTOR and activation of LC3I/II in HepG2 cells and mice livers. Treatment of HepG2 cells with the ROS scavenger N-acetyl-l-cysteine alleviated ER stress stimulation and induced HepG2 cell death. Moreover, suppression of autophagy with 3-Methyladenine enhanced HepG2 cells apoptosis. Concurrently, four consecutive days exposure of mice livers to FB1 altered the levels of sphingoid bases, hepatic enzymes and induced histopathological changes. Moreover, the expression levels of major ER stress and autophagy-related markers such as PERK, IRE1-α, and LC3I/II also increased. Autophagy activation protected HepG2 cells and mice livers from the lethal effects of FB1. Hence, these findings specify that, the compounds that modify autophagy might be useful therapeutic agents for treatment of patients with FB1 induced liver ailments.

Interruption of Inositol Sphingolipid Synthesis Triggers Stt4p-dependent Protein Kinase C Signaling

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.

Exposure to dioxin-like carcinogens of the population of the Canary Islands (Spain) through the intake of dairy products

Fumonisins (FB) are mycotoxins produced by Fusarium verticillioides, frequently associated with corn. It produces speciesspecific and organ-specific toxicity, including teratogenicity, equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. FB interferes with ceramide synthase leading to intracellular accumulation of sphingoid bases which mediate several key biological processes such as cell proliferation, and DNA replication, inhibits biosynthesis of cellularmacromolecules and induces lipidperoxidation in rathepatocytes. The current studywas conducted to evaluate theprotective effect of the ethanol extract of jojoba in rats fed FB-contaminated diet. Forty mature male Sprague–Dawley were divided into four treatment groups and treated for 3 weeks as follow: the control group, the group fed FB-contaminated diet (150mg/kg), the group treated orally with jojoba extract (5mg/kg b.w.) and the group fed FB-contaminated diet and treated with the extract. At the end of treatment period, blood and liver samples were collected for heamatological, biochemical andhistological studies. The results indicated that FB induced significant decrease in red blood cells count, hemoglobin concentration, platelets, white blood cells count, cholesterol and total antioxidant capacity (TAC) and a significant increase in ALT, AST, ALP, LDH, CK, urea, createnine, uric acid and lipid peroxidation (MDA) accompanied with severe pathological changes in the liver tissues. Animals treated with jojoba extract alone were comparable to the controls regarding the heamatological and the biochemical parameters and the histological picture of the liver. The extract contracted the toxic effects of FB and succeeded to normalize all the parameters tested and the histological picture of the liver.

Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.

A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings

The fungus Fusarium verticillioides is a pathogen of many plants and produces fumonisins. In addition to their well-studied animal toxicoses, these toxins contribute to the development of maize seedling disease in susceptible maize varieties. Fumonisin disruption of sphingolipid biosynthesis occurs during pathogenesis. An extraction method was developed for the simultaneous analysis of fumonisins B(1) (FB(1)), B(2) (FB(2)) and B(3) (FB(3)), free sphingoid bases and sphingoid base 1-phosphates in maize tissues by liquid chromatography/linear ion trap tandem mass spectrometry. The method involved a single extraction using 1:1 acetonitrile:water + 5% formic acid (1 ml per 10 mg tissue). Mean recoveries ranged from approximately 50 to 99 percent, and limits of detection ranged from 10 fg microl(-1) to 6900 fg microl(-1). To test the efficacy of the method, seeds of a susceptible maize line were inoculated with a pathogenic, fumonisin-producing strain of F. verticillioides. The seedlings were then harvested, and fumonisin content, as well as sphingoid bases and their 1-phosphates, were measured in the leaf and root tissues. Fumonisin accumulation was significantly greater in leaf one compared to leaves two and three. While FB(1), FB(2), and FB(3) were detected in root tissues, FB(1) was preferentially accumulated in leaf tissues. Accumulation of sphingoid bases and their 1-phosphates was evident in roots and leaves of seedlings grown from inoculated seed, with the level of accumulation being similar in leaves 1, 2 and 3. The method developed was effective, fast, and sensitive for use in simultaneously measuring fumonisin in tissues and their effects on sphingolipid metabolite biomarkers of disease. The method should be useful for screening maize cultivars for susceptibility to F. verticillioides-induced seedling diseases.

Inducible nitric oxide has protective effect on fumonisin B1 hepatotoxicity in mice via modulation of sphingosine kinase

Fumonisin B1, a mycotoxin, is an inhibitor of ceramide synthase causing marked dysregulation of sphingolipid metabolism in cells. This mycotoxin causes accumulation of free sphingoid bases (sphingosine and dihydrosphingosine or sphinganine) and their metabolites, important messengers involved in signal transduction leading to either cell survival or death. Free sphingoid bases are known apoptotic molecules whereas sphingosine 1-phosphate is protective. We previously reported that fumonisin B1 caused sphingosine kinase (SPHK) induction along with the increase of serine palmitoyltransferase (SPT). Fumonisin B1 also increased inducible nitric oxide synthase (iNOS) expression. In the current study we employed a mouse strain with the targeted deletion of iNOS gene (Nos-KO) to evaluate the role of nitric oxide (NO) on fumonisin B1-induced hepatotoxicity. The Nos-KO mice exhibited increased hepatotoxicity after subacute fumonisin B1 exposure compared to their wild type counterparts, the liver regeneration was lower in Nos-KO compared to that in the WT mice. Increased hepatotoxicity in Nos-KO was not related to the extent of free sphingoid base accumulation after fumonisin B1 treatment; however, it was accompanied by a lack of fumonisin B1-induced SPHK induction. The fumonisin B1-induced SPT was unaffected by lack of iNOS gene. Deletion of iNOS gene did not prevent fumonisin B1-dependent induction of inflammatory cytokines, namely tumor necrosis factor α, interferon γ and interleukin-12. The lack of fumonisin B1-induced SPHK induction in Nos-KO was supported by a similar effect on phosphorylated metabolites of sphingoid bases; the equilibrium between sphingoid bases and their phosphates is maintained by SPHK. We therefore conclude that iNOS induction produced by fumonisin B1 modulates SPHK activity; the lack of iNOS prevents generation of sphingosine 1-phosphate and deprives cells from its protective effects.

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B(1) is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B(1) caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B(1) induced hepatic apoptosis in mice. The role of tumor necrosis factor alpha (TNFalpha) in fumonisin B(1) mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B(1) induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B(1) caused accumulation of sphingoid bases and led to increase in TNFalpha expression. Phosphorylated and total c-Jun NH(2)-terminal kinase (JNK) activities were increased after 24 h fumonisin B(1) treatment. JNK inhibitor (SP600125) and anti-TNFalpha reduced the apoptosis induced by fumonisin B(1). The role of JNK signaling in fumonisin B(1) induced apoptosis is downstream of TNFalpha production, as fumonisin B(1)-mediated activation of JNK was reduced by the presence of anti-TNFalpha in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B(1) induced TNFalpha expression. Results of this study imply that generation of fumonisin B(1) induced TNFalpha results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes.

Myriocin prevents fumonisin B 1-induced sphingoid base accumulation in mice liver without ameliorating hepatotoxicity

Fumonisin B 1 (FB 1), a mycotoxin produced by Fusarium verticillioides present on corn and corn-based products, causes species- and organ-specific diseases. The hepatotoxic effects of FB 1 in mice have been closely correlated with the accumulation of free sphinganine, a marker for ceramide synthase inhibition, and reduced biosynthesis of more complex sphingolipids. It has been shown that FB 1 modulates expression of many cell signaling factors. In the current study we used myriocin, a specific inhibitor of serine palmitoyltransferase, to investigate the role of free sphinganine accumulation in FB 1-induced hepatotoxicity and increased expression of selected signaling genes in BALB/c mice. The mice were pretreated daily with intraperitoneal injection of 1.0 mg/kg myriocin 30 min before subcutaneous injections of 2.25 mg/kg of FB 1 for 3 days. Results showed that myriocin alone was not hepatotoxic and the combination of myriocin plus FB 1 completely prevented the FB 1-induced elevation of hepatic free sphinganine and prevented the FB 1-induced induction of selected cell signaling genes, suggesting that accumulation of free sphinganine and/or its metabolites contribute to the FB 1-modulation of the cell signaling factors. However, the combination of myriocin and FB 1 did not prevent FB 1-increased concentration of plasma alanine aminotransferase and only slightly attenuated aspartate aminotransferase; it did not affect the FB 1-induced hepatocyte apoptosis or increased cell proliferation. A longer combined treatment of myriocin and FB 1 was highly toxic. The hepatotoxic effects in mice seen in this study are most likely due to a combination of factors including accumulation of free sphinganine, depletion of more complex sphingolipids and sphingomyelin, or other unknown mechanisms.

Digestion of Maize Sphingolipids in Rats and Uptake of Sphingadienine by Caco-2 Cells

We investigated the digestion of cerebrosides of plant origin prepared from maize, focusing especially on the digestive fates of trans-4, cis-8- and trans-4, trans-8-sphingadienine, which are common in higher plants. In the small intestinal mucosa and cecal contents of rats, the cerebrosidase activity at pH 5.2 toward the glucosyl linkage in maize cerebrosides (glucosylceramides) was similar to that in cerebrosides of mammalian origin. Similarly, the ceramidase activity toward the amide linkage in ceramides prepared from maize cerebrosides at pH 7.0 was the same as that toward ceramides of mammalian origin. In addition, maize cerebrosides were hydrolyzed to ceramide and free sphingoid bases in the digestive tract of rats after oral administration. To further evaluate the uptake by enterocytes of 4,8-sphingadienine, we used differentiated Caco-2 cells, derived from human colonic carcinoma, as a model of intestinal epithelial cells. The accumulation of sphingoid bases in Caco-2 cells incubated with each isomer of sphingadienine was lower than that after incubation with sphingosine (P < 0.05). Verapamil, a P-glycoprotein inhibitor, increased the accumulation of each sphingadienine but not of sphingosine, suggesting that the efflux of sphingadienine of plant origin, but not sphingosine of mammalian origin, was affected by P-glycoprotein. The digestibility of maize cerebrosides appears similar to that of cerebrosides of mammalian origin, but the metabolic fate of sphingoid bases of plant origin within enterocytes differs from that of sphingosine. Isomers of 4,8-sphingadienine degraded from dietary plant cerebrosides appear to be poorly absorbed from the digestive tract.

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

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 B(1) (FB(1)), (2) the minimum threshold dose of FB(1) 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 FB(1), 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 FB(1)/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 FB(1) followed by daily exposure at low levels will prolong the elevation of sphinganine in kidney. In cultured renal cells FB(1) 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.

Fumonisin-Induced Tumor Necrosis Factor-α Expression in a Porcine Kidney Cell Line Is Independent of Sphingoid Base Accumulation Induced by Ceramide Synthase Inhibition

Previous studies have shown that fumonisin B1 (FB1) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-α (TNFα) plays an important role in FB1 toxicity and the expression of TNFα mRNA in liver and kidney is increased following FB1 exposure in mice. The objective of the current study was to investigate whether these two events (sphingoid bases accumulation and TNFα induction) are dependent on each other. An increase in expression of TNFα mRNA was detected in LLC-PK1 cells as early as 4 h after FB1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNFα mRNA and FB1 concentration. Increases of intracellular sphingoid bases were also detected after 4 h of FB1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNFα. Inhibition of sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free sphingoid bases in response to FB1, but it did not prevent the induction of TNFα expression. Results indicate that FB1-induced increase in TNFα expression is independent of sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK1 cells.

Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins.

Sphingolipids have important roles in membrane and lipoprotein structure and in cell regulation as second messengers for growth factors, differentiation factors, cytokines, and a growing list of agonists. Bioactive sphingolipids are formed both by the turnover of complex sphingolipids and as intermediates of sphingolipid biosynthesis. Usually, the amounts are highly regulated; however, by inhibiting ceramide synthase, fumonisins block the biosynthesis of complex sphingolipids and cause sphinganine (and sometimes sphingosine) to accumulate. Where the mechanism has been studied most thoroughly, the accumulation of sphingoid bases is a primary cause of the toxicity of fumonisin B (FB). Nonetheless, the full effects of fumonisins probably involve many biochemical events. The elevations in sphingoid bases also affect the amounts of other lipids, including the 1-phosphates and N-acetyl derivatives of sphinganine. Furthermore, the aminopentol backbone of FB1 (AP1) is both an inhibitor and a substrate for ceramide synthase, and the resultant N-palmitoyl-AP1 (PAP1) is an even more potent inhibitor of ceramide synthase (presumably as a product analog). PAP1 is 10 times more toxic than FB1 or AP1 for HT-29 cells in culture, and hence may play a role in the toxicity of nixtamalized fumonisins. All these processes--the effects of fumonisins on sphingolipid metabolism, the pathways altered by perturbation of sphingolipid metabolism, and the complex cellular behaviors regulated by sphingolipids--must be borne in mind when evaluating the pathologic effects of fumonisins.

Tolerance to fumonisin toxicity in a mouse strain lacking the P75 tumor necrosis factor receptor

Fumonisin B 1 (FB 1), a potent mycotoxin prevalent in corn and cereals, causes a variety of toxic effects in different mammalian species. The biochemical responses of FB 1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a possible involvement of tumor necrosis factor α (TNFα). To further characterize the role of TNFα, toxic response to FB 1 was investigated in male C57BL/6J mice (WT) and a corresponding TNFα receptor knockout (TRK) strain, genetically modified to lack the TNFα1b receptor. The hepatotoxic effects of 5 daily injections of 2.25 mg/kg per day of FB 1 were observed in WT but were reduced in TRK, evidenced by circulating alanine aminotransferase and aspartate aminotransferase levels and histopathological evaluation of the tissue. FB 1 induced TNFα expression in the livers of both WT and TRK mice to a similar extent (3–4 fold over control); however, a corresponding increase of cellular NFκB, expected after the downstream cellular signaling of TNFα, was noted only in the WT. Accumulation of liver sphingosine after FB 1 treatment was similar in both WT and TRK, but the FB 1-induced increases in liver sphinganine and kidney sphingosine and sphinganine were lower in TRK than in WT. Results emphasized the role of TNFα in FB 1-induced hepatotoxicity in mice and the possible relationship of sphingoid base accumulation and TNFα induction. Moreover, the presence of TNFα receptor 1b appears to be important in mediating the hepatotoxic responses of TNFα and FB 1 in mice.

Serine palmitoyltransferase inhibition reverses anti-proliferative effects of ceramide synthase inhibition in cultured renal cells and suppresses free sphingoid base accumulation in kidney of BALBc mice

The purpose of this study was to determine the ability of the fungal serine palmitoyltransferase (SPT) inhibitor, myriocin, to prevent the anti-proliferative and cytotoxic effects of fumonisin B 1 in cultured pig kidney epithelial cells, LLC-PK 1. In an earlier study with LLC-PK 1 cells, β-chloroalanine (a nonspecific SPT inhibitor) was found to inhibit the fumonisin-induced accumulation of free sphinganine by >90% but only partially reversed (50–60%) fumonisin’s antiproliferative and cytotoxic effects. β-Chloroalanine is not the ideal SPT inhibitor for this type of study because it also inhibits other pyridoxal 5′-phosphate-dependent enzymes. A potent and selective fungal SPT inhibitor (myriocin) was partially purified from liquid cultures of Isaria (= Cordyceps) sinclairii by a combination of organic extraction and column chromatography. The various fractions were bioassayed for their ability to inhibit fumonisin-induced sphinganine accumulation in LLC-PK 1 cells. The activity in partially purified material was compared to the activity of highly purified myriocin and the results expressed as myriocin equivalents. The estimated IC 50 and IC 95 for inhibition of fumonisin-induced sphinganine accumulation were approximately 1.8 and 22 nM, respectively. The IC 95 concentration of the fungal SPT inhibitor reversed the antiproliferative effects and prevented fumonisin-induced apoptosis after 48 h exposure to 50 μM fumonisin B 1. The SPT inhibitor was also effective at reducing free sphinganine in vivo. Free sphinganine concentration was reduced 60% in kidney of mice injected i.p. with SPT inhibitor plus fumonisin B 1 when compared to fumonisin B 1 alone. The ability of SPT inhibition to reduce fumonisin B 1-induced sphinganine accumulation in vivo may be useful in the development of therapeutic agents for treatment of animals suspected to have been exposed to toxic levels of fumonisin in feeds.

Disruption of sphingolipid metabolism and stimulation of DNA synthesis by fumonisin B1. A molecular mechanism for carcinogenesis associated with Fusarium moniliforme.

Consumption of grains contaminated with Fusarium moniliforme (Sheldon) causes liver cancer in rats and has been correlated with esophageal cancer in humans. The causative agents are believed to be a family of compounds known as fumonisins, which bear remarkable structural resemblances to sphingosine and sphinganine, the long-chain (sphingoid) base backbones of sphingolipids. Recently, fumonisin B1 has been shown to block de novo synthesis of sphingolipids by inhibiting sphingosine (sphinganine) N-acyltransferase, which leads to accumulation of sphingoid bases. Because the exogenous addition of sphingosine and sphingosine 1-phosphate to Swiss 3T3 cells has been shown to stimulate DNA synthesis (Zhang, H., Buckley, N.E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81; Zhang, H., Desai, N.N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167), we hypothesized that fumonisins might stimulate DNA synthesis by disrupting sphingolipid metabolism. Fumonisin B1 caused accumulation of sphinganine and sphingosine in Swiss 3T3 fibroblasts and, as occurred when these sphingoid bases were added exogenously, stimulated thymidine incorporation into DNA and augmented the mitogenic effect of insulin in a concentration-dependent manner. The mechanism underlying the mitogenic effect of fumonisin B1 was further investigated by using beta-fluoroalanine to block the initial step of sphingolipid biosynthesis catalyzed by serine palmitoyltransferase. beta-Fluoroalanine reduced sphingoid base accumulation in fumonisin B1-treated fibroblasts and inhibited fumonisin B1-stimulated DNA synthesis, but had no effect on mitogenesis when added alone. Fumonisin B1 did not cause accumulation of sphinganine 1-phosphate; therefore, it appears that sphingoid bases per se can stimulate DNA synthesis. To prove that the 1-phosphate is not obligatory, a 1-deoxysphinganine was synthesized, and it was as potent as sphinganine in stimulating DNA synthesis. These results establish that fumonisin B1 is mitogenic via accumulation of sphingoid bases rather than inhibition of complex sphingolipid biosynthesis per se. Because mitogens can often affect cell transformation, this provides a plausible molecular mechanism to explain the carcinogenicity of fumonisins.