Fumonisin inhibition of de Novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK 1 cells

Abstract

Fumonisins are a group of structurally related compounds produced by Fusarium moniliforme. Recently, it has been shown that fumonisins B 1 and B 2 are the first naturally occurring inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase) in rat primary hepatocytes (Wang et al. J. Biol. Chem. 266, 14,486–14,490, 1991). These enzymes are key components in the pathways for de novo sphingolipid biosynthesis and sphingolipid turnover. The results of the present study show that fumonisins B 1 and B 2 inhibit proliferation and are cytotoxic to LLC-PK 1 cells. Concentrations of fumonisin B 1 and B 2 between 10 and 35 μ m inhibited cell proliferation, whereas higher concentrations (>35 μ m) killed cells. Inhibition of cell proliferation and cell death were preceded by a lag period of at least 24 hr during which cells appeared to be functioning normally. Cells exposed to fumonisin B 1 exhibited normal growth kinetics and morphology soon after fumonisin B 1 was removed; thus, the effects of fumonisin B 1 were reversible. The EC 50 for alterations in sphingolipid biosynthesis was 10 to 15 μ m. Inhibition of de novo sphingolipid biosynthesis occurred before inhibition of cell proliferation or cytotoxicity, and the dose response for the decrease in the [ 3H]sphingosine to [ 3H]sphinganine ratio at 7 hr closely paralleled the dose response for effects on proliferation and cytotoxicity at 3–5 days. In addition, the level of free sphinganine, and to a lesser extent sphingosine, increased in fumonisin-treated cells in a dose-dependent manner. During the 24-hr lag period preceding inhibition of cell proliferation, the free sphinganine content increased by 12,800% in cells exposed to 35 μ m fumonisin B 1. Whereas a mechanistic relationship between the inhibition of de novo sphingolipid biosynthesis and inhibition of proliferation and cell death has not been demonstrated, the results of this study support the hypothesis that inhibition of de novo sphingolipid biosynthesis is an early event in the toxicity of fumonisins to LLC-PK 1 cells.