Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine: A NOVEL CATEGORY OF BIOACTIVE 1-DEOXYSPHINGOID BASES AND 1-DEOXYDIHYDROCERAMIDES BIOSYNTHESIZED BY MAMMALIAN CELL LINES AND ANIMALS*

Fumonisin B₁ (FB₁) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB₁ increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C₁₈H₄₀NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH₃CH=NH ⁺₂) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-¹³C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK₁ cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-¹³C]palmitate into 1-[¹³C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[¹³C]deoxySa. 1-DeoxySa was elevated in FB₁-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK₁ and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB₁, substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and “ceramides” that might play important roles in cell regulation.Fumonisins (FB)² cause diseases of horses, swine, and other farm animals and are regarded to be potential risk factors for human esophageal cancer (1) and, more recently, birth defects (2). Studies of this family of mycotoxins, and particularly of the highly prevalent subspecies fumonisin B₁ (FB₁) (reviewed in Refs. 1 and 2), have established that FB_1, is both toxic and carcinogenic for laboratory animals, with the liver and kidney being the most sensitive target organs (3, 4). Other FB are also toxic, but their carcinogenicity is unknown.FB are potent inhibitors of ceramide synthase(s) (CerS) (5), the enzymes responsible for acylation of sphingoid bases using fatty acyl-CoA for sphingolipid biosynthesis de novo and recycling pathways (6). As a consequence of this inhibition, the substrates sphinganine (Sa) and, usually to a lesser extent, sphingosine (So), accumulate and are often diverted to sphinganine 1-phosphate (Sa1P) and sphingosine 1-phosphate (S1P), respectively (7), while the product N-acylsphinganines (dihydroceramides), N-acylsphingosines (ceramides, Cer), and more complex sphingolipids decrease (5, 7). This disruption of sphingolipid metabolism has been proposed to be responsible for the toxicity, and possibly carcinogenicity, of FB, based on mechanistic studies with cells in culture (5, 7–9). This has been borne out by a number of animal feeding studies that have correlated the elevation of Sa in blood, urine, liver, and kidney with liver and kidney toxicity (4, 7, 10, 11).Most of the mechanistic studies have focused on the accumulation of free Sa and other sphingoid bases, because these compounds are highly cytotoxic, although the large number of bioactive metabolites in this pathway make it likely that multiple mediators may participate (7, 9). Nonetheless, inhibition of serine palmitoyltransferase (SPT), the initial enzyme of de novo sphingolipid biosynthesis, reverses the increased apoptosis and altered cell growth induced by FB₁ treatment (12–19). Therefore, it is likely that these effects of FB₁ are due to the accumulation of cytotoxic intermediate(s) rather than depletion of downstream metabolites, because the latter also occurs when SPT is inhibited.In studies of the effects of FB₁ on the renal cell line LLC-PK₁ (20),³ we have noted that in addition to the elevation of Sa and So, there is a large increase in an unidentified species that appears to be a sphingoid base, because it is extracted by organic solvents, derivatized with ortho-phthalaldehyde (OPA), and eluted from reverse-phase liquid chromatography (LC) in the sphingoid base region. Herein we report: (i) the isolation and characterization of this novel sphingoid base as 1-deoxysphinganine (1-deoxySa); (ii) that its origin is the utilization of alanine instead of serine by SPT as well as that the N-acyl-derivatives of 1-deoxySa (1-deoxydihydroceramides (1-deoxyDHCer)) are normally found in mammalian cells; (iii) that 1-deoxySa has cytotoxicity comparable to other sphingoid bases elevated by FB₁; and (iv) that 1-deoxySa is not only elevated in cells in culture but also in tissues of animals exposed to dietary FB and, therefore, might contribute to diseases caused by these mycotoxins.