Amplification and expression of the TGF-α, EGF receptor and c-myc genes in four human oesophageal squamous cell carcinoma lines

We have previously shown that four human oesophageal squamous cell carcinoma (SCC) cell lines secrete significant quantities of transforming growth factor alpha (TGF-)in vitro. Three of these lines are known to produce supernumary low-affinity epidermal growth factor receptors (EGF-Rs). Using an¹²⁵I-EGF compeitive binding assay and Scatchard analysis, we show that the fourth also overproduces low-affinity receptors. According to slot blot DNA analyses, the secretion of high levels of TGF- is not associated with amplification of the TGF- gene, and hyperproduction of the EGF-R is correlated with receptor gene amplification. Western blot analyses show that the c-Myc protein is overexpressed in two of the cell lines; and Southern and Northern blot analyses indicate that this overexpression occurs independently of c-myc gene amplification. Our results are consistent with an autocrine role for TGF- and EGF-R in oesophageal carcinogenesis and support the possibility that c-myc overexpression may be required for thein vivo tumourigenicity of cells that produce high levels of TGF- and the EGF-R.     

Cell Polarity Factor Par3 Binds SPTLC1 and Modulates Monocyte Serine Palmitoyltransferase Activity and Chemotaxis

Elevated sphingolipids have been associated with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and -2 subunits, and here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein Par3 (partitioning defective protein 3). Mammalian SPTLC1 orthologs have a highly conserved C terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with an SPTLC1 C-terminal peptide, we found it bound the third PDZ domain of Par3. Overlay and immunoprecipitation assays confirmed this interaction and indicate Par3 is able to associate with the SPTLC1/2 holoenzyme by binding the C-terminal SPTLC1 PDZ motif. The physiologic existence of the SPTLC1/2-Par3 complex was detected in mouse liver and macrophages, and short interfering RNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and de novo ceramide synthesis by nearly 40%. Given monocyte recruitment into inflamed vessels is thought to promote atherosclerosis, and because Par3 and sphingolipids have been associated with polarized cell migration, we tested whether the ability of THP-1 monocytes to migrate toward MCP-1 (monocyte chemoattractant protein 1) depended upon Par3 and SPTLC1 expression. Knockdown of Par3 significantly reduced MCP1-induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity and was blunted by ceramide treatment. In conclusion, protein arrays were used to identify a novel SPTLC1-Par3 interaction that associates with increased monocyte serine palmitoyltransferase activity and chemotaxis toward inflammatory signals.Sphingolipids are a structurally diverse class of lipids that play correspondingly diverse roles in membrane structure, cell proliferation, immune function, and skin physiology (1–4). De novo sphingolipid synthesis is initiated by serine palmitoyltransferase (SPT),² an enzyme that condenses serine and palmitoyl-CoA forming the biosynthetic intermediate 3-ketodihydrosphingosine that is subsequently converted to ceramide, sphingomyelin, and other sphingolipids (5). SPT is a heterodimer composed of the SPTLC1 and -2 subunits (6, 7), which may form higher order multimeric structures that can include a third subunit, SPTLC3 (8, 9). Both the SPTLC2 and -3 subunits are catalytically active and contain conserved lysines that act as Schiff bases during the condensation reaction (5, 8). In contrast, SPTLC1 does not contain the conserved catalytically active lysine, but is important for stabilizing the SPTLC2 subunit and anchoring the SPT holoenyzme on the cytosolic face of the endoplasmic reticulum (10, 11).Expression and regulation of the SPTLC1/2 holoenyzme are of interest because its activity controls de novo synthesis of sphingomyelin, and increased plasma levels of this sphingolipid have been correlated with an increased incidence of cardiovascular disease in humans (12, 13). Conversely, inhibition of SPT activity with myriocin, a fungal metabolite, strongly inhibits atherosclerotic development in ApoE^−/− mice (14–18). Moreover, the increased atherosclerosis seen in ApoE^−/− mice has been associated with a post-translational increase in liver SPT activity (19). How SPT activity and sphingolipids may act to promote the progression of atherosclerosis is unclear, but the data do suggest analysis of factors that regulate SPT activity should provide mechanistic insight into the link between de novo sphingolipid synthesis and atherosclerosis. In this regard we have found that SPTLC1 can interact with the ABCA1 transporter and inhibit its ability to transfer cholesterol to apoA-I, a mechanism that would be expected to promote atherosclerosis (20). Thus, along with playing a direct role in the synthesis of sphingolipids, SPTLC1 may also have evolved as an SPT subunit whose function is to regulate SPT activity in response to the cellular demand for sphingolipids and other membrane constituents such as cholesterol. To play such a role, SPTLC1 may engage additional protein-protein interactions that integrate input from signaling pathways and allow SPTLC1 to modulate SPT activity in response to altered demand for sphingolipids.Here we have explored this hypothesis by first conducting a protein array screen for SPTLC1 interacting factors. Consistent with the potential to engage cellular factors in protein-protein interactions, sequence alignment of the SPTLC1 C terminus indicates it has been strongly conserved in mammals as a type II PDZ domain binding motif. Moreover, because topology studies indicate the SPTLC1 C terminus resides in the cytoplasm where it could be bound by PDZ proteins, we used protein arrays spotted with 123 PDZ domains from 73 different proteins to screen for interactions with the SPTLC1 C terminus. This screen indicated the SPTLC1 C terminus directly interacts with the third PDZ domain of PARD3 (partitioning defective protein 3). PARD3, also known as Par3, is a scaffolding factor that recruits signaling molecules, including atypical protein kinase C and Cdc42 into multiprotein complexes that regulate formation of membrane microdomains required for apical/basal polarity and for directed cell migration (21–25). Mutation analysis confirmed the SPTLC1-Par3 interaction depended upon the SPTLC1 C-terminal PDZ motif, and immunoprecipitation assays indicate Par3 is able to associate with the SPTLC1/2 holoenyzme by binding the SPTLC1 C-terminal PDZ motif. The Par-3 interaction with the SPTLC1/2 holoenyzme was detected in the liver, a major site of SPT activity and sphingolipid synthesis. Given SPT activity is proatheroslerotic, and because we have also detected SPTLC1 expression in macrophages, a cell type that plays a central role in the progression of atherosclerosis, we tested and found that Par3 was expressed and interacted with the SPT holoenyzme in primary mouse macrophages. Significantly, loss of Par3 expression in human THP-1 monocyte macrophages reduced SPT activity and inhibited their ability to migrate toward MCP-1 (monocyte chemotactic protein-1). Likewise, shRNA suppression of SPTLC1 reduced monocyte migration toward MCP-1, as did myriocin inhibition of SPT activity, an effect that was blunted by loss of Par3 expression. In aggregate, our work has identified a novel protein-protein interaction between SPTLC1 and Par3 that is associated with an increase in SPT activity and the promotion of polarized cell migration in response to an inflammatory signal.     

Effects of Inhibition of Serine Palmitoyltransferase (SPT) and Sphingosine Kinase 1 (SphK1) on Palmitate Induced Insulin Resistance in L6 Myotubes

Background

The objective of this study was to examine the effects of short (2 h) and prolonged (18 h) inhibition of serine palmitoyltransferase (SPT) and sphingosine kinase 1 (SphK1) on palmitate (PA) induced insulin resistance in L6 myotubes.

Methods

L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor) or PA and Ski II (SphK1inhibitor) for different time periods (2 h and 18 h). Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA), ceramide (CER), sphingosine (SFO), sphingosine-1-phosphate (S1P)] were estimated by HPLC.

Results

Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes.

Conclusion

Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor). Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.     

The genomic structure of the proto-oncogene c-kit encoded at the murineWhite spotting locus

The proto-oncogene c-kit encodes a transmembrane receptor with tyrosine kinase activity, which transduces signal fromkit ligand (KL), and is responsible for hematogenesis, melanogenesis and gametogenesis during fetal development and adult life. Partial or complete loss of c-kit function due to mutation of the c-kit or KL gene accounts for the phenotypes of the murineWhite-spotting andSteel mutations, respectively. The c-kit protein has the structural features of extracellular immunoglobulin-like domains and intracellular kinase domain with a hydrophilic insert. These features have categorized c-kit along with platelet-derived growth factor receptors, colony-stimulating factor 1 receptor (c-fms) and others to subclass III of the receptor tyrosine kinases. We report the structure of the murine c-kit gene. The c-kit gene consists of 21 exons and spans at least 70 kb. The 5 and 3 flanking exons encode the untranslated sequences as well as part of the coding sequence. The internal exons are typically small with each of them encoding a structurally important subunit of the protein. Comparison of gene structures of members of the subclass III receptor tyrosine kinases has improved our understanding of the structure-functional relationship of the c-kit protein.     

Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines

Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two early replicating (i.e., active) X chromosomes and lost the late replicating (i.e., inactive) X.     

Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells

In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that theras product is able to transactivate the -actin, CMV and SR promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with theras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify theras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

Cadmium induces hypertrophy accompanied by increased myc mRNA accumulation in NRK-49F cells

Previous studies showed that Cd⁺⁺ inhibits EGF-induced DNA synthesis that not EGF-induced myc mRNA accumulation or amino acid incorporation into protein in serum-starved NRK-49F cells. In this study, flow cytometry was used to analyze the DNA and protein content of individual cells stimulated with Cd⁺⁺ and/or epidermal growth factor (EGF). myc oncogene expression in these cells was also measured. It was found that, in both parental NRK-49F cells and in a clonal subpopulation, N1, Cd⁺⁺ induces an hypertrophic response. In parental NRK-49F cells, however, lower doses of Cd⁺⁺ (0.5 M) induced more pronounced hypertrophic responses than did higher doses (4 M); whereas in N1 cells, the Cd⁺⁺-induced hypertrophic response shows a pattern of increasing response with doses of Cd⁺⁺ from 0.5 to 4 M. myc mRNA accumulation measured 2 hours after stimulation correlated with the hypertrophic responses in both NRK-49F cells and in N1 cells. The results show that Cd⁺⁺-induced hypertrophy in NRK-49F cells is associated with increased myc oncogene mRNA accumulation, indicating that cell proliferation and cell hypertrophy may in part share common activation pathways.Abbreviations EGF Epidermal growth factor - FCM Flow cytometric analysis - FITC fluorescein isothiocyanate - PI propidium iodide     

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

Background

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Methods

The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.

Results

We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.

Conclusions

The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.

General significance

Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.     

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.     

Effects of oxygen free radicals on articular chondrocytes in culture: C-myc and c-Ha-ras messenger RNAs and proliferation kinetics

Since oxygen free radicals are believed to play an important role in cartilage degradation, we studied the effects of these radicals generated by the hypoxanthine-xanthine oxidase system on rabbit articular chondrocytes in culture. Among the damages induced by these radicals, cell proliferation inhibition and G2 arrest were observed. To elucidate the mechanisms involved in this phenomenon, the expression of c-myc and c-Ha-ras genes whose products are associated with cell growth control was studied. Results showed that in chondrocytes, c-myc and c-Ha-ras expression was particularly important during the G1 phase of the cell cycle and that oxygen reactive species, especially H₂O₂, induced an important decrease of c-myc and c-Ha-ras mRNA levels. Chondrocytes cell cycle analysis revealed an accumulation of cells in G2 phase. It led us to suggest that the chondrocyte cell cycle perturbations observed after oxygen free radicals treatment could be associated with the decrease of c-myc and c-Ha-ras expression.     

The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C₁₆-sphinganine and C₁₆-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C₁₆-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C₁₆ backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C₁₆-sphingoid bases and that sphingolipids with a C₁₆ backbone constitute a significant proportion of human plasma sphingolipids.Sphingolipids comprise a class of bioactive lipids that contribute to plasma membrane and plasma lipoprotein formation and exert a broad range of cellular signaling functions such as cell proliferation, endocytosis, and the response of cells to inflammatory and apoptotic stress signals (1–4).Sphingolipids are derived from the aliphatic amino alcohol sphingosine, which is formed from the precursors l-serine and palmitoyl-CoA. The condensation of serine with palmitoyl-CoA is catalyzed by the enzyme serine palmitoyltransferase (SPT)³ (EC 2.3.1.50) and leads to the intermediate 3-ketodihydrosphingosine. 3-Ketodihydrosphingosine is then rapidly converted to dihydrosphingosine (sphinganine) and dihydroceramide. The desaturation of dihydroceramide generates ceramide, and the breakdown of ceramide by ceramidase finally forms sphingosine. The sphingosine backbone of ceramide is usually O-linked to a polar head group such as phosphocholine or carbohydrates and amide-linked to an acyl group. The combination of the sphingosine backbone with different head groups, in particular with various oligosaccharides, leads to a complex variety of different sphingolipid metabolites (5, 6). Moreover, it was shown recently that SPT is also able to use l-alanine as an alternative substrate, thereby generating the atypical sphingoid base 1-deoxysphinganine (7).SPT belongs to the family of pyridoxal phosphate-dependent α-oxoamine synthases. Other members of this family include 5-aminolevulinic acid synthase, 2-amino-3 ketobutyrate ligase, and 8-amino-7-oxononanoate synthase (8). SPT is ubiquitously expressed, and enzyme activity has been detected in all tissues tested so far including brain, lung, liver, kidney, and muscle (9). SPT is essential for embryonic development, and homozygous SPT knock-out mice are not viable (10). SPT has been believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. The two subunits SPTLC1 and SPTLC2 show a similarity at AA level of ∼20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, only the SPTLC2 subunit contains a pyridoxal phosphate binding motif (8, 11).Recently, we identified and cloned a novel third SPT subunit (SPTLC3) (12). The SPTLC3 sequence shows 68% homology to the SPTLC2 subunit and also includes a pyridoxal phosphate consensus motive. The SPTLC3 gene is present in mammals, birds, and some lower vertebrates like fish (Danio rerio) and frog (Xenopus laevis) but not in invertebrate lineages. The SPTLC3 mRNA has been detected in most human tissues with a particularly high expression in placenta (12), indicating a special role for SPTLC3 during development and pregnancy. By using immunoprecipitation, native gel analysis, cross-linking studies, and size exclusion chromatography, it was demonstrated that the native SPT enzyme contains all three subunits and forms a protein complex with a molecular mass of about 460 kDa (13). However, because SPTLC2 and SPTLC3 are encoded by two distinct genes and expressed within the same cell types, we assume a distinct function for the two subunits. One of these differences might be altered substrate affinity or enzymatic activity. This issue is addressed in the present study.     

Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c mice

Fumonisin B₁ is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B₁ toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B₁ on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B₁ subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B₁ treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 281–289, 1998