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Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival. J. Nutr. 131: 2826-2830, 2001. KEY WORDS: * C3H10T1/2 cells * ceramide * sphingosine-1-phosphate * apoptosis

Fumonisin [B.sub.1] (F[B.sub.1]) is a frequently encountered mycotoxin that inhibits ceramide synthase, the enzyme that acylates sphinganine, sphingosine and other 'sphingoid' bases. Exposure of rats, rabbits, pigs and nonhuman primates to fumonisin-contaminated feed elevates sphingoid base amounts in urine; therefore, this study examined the time course and reversibility of these changes. When an AJN-76 diet supplemented with [greater than or equal to] 5 [Mu]g F[B.sub.1]/g was fed to male Sprague-Dawley rats, there was a significant increase in sphinganine (ca. 50-fold in urine from rats fed 50 [Mu]g F[B.sub.1]/g diet) and smaller changes in sphingosine within 5 to 7 d, compared to rats fed the same diet without F[B.sub.1]. No change occurred in sphingoid bases upon feeding 1 [Mu]g F[B.sub.1]/g for up to 60 d. When rats were fed F[B.sub.1] (10 [Mu]g F[B.sub.1]/g diet for 10 d), then changed to the same diet minus F[B.sub.1], urinary sphingoid bases returned to normal within 10 d. However, if the rats were fed 10 [Mu]g F[B.sub.1]/g for 10 d, then changed to 1 [Mu]g F[B.sub.1]/g, the amounts of sphingoid bases in urine were the same as for rats that were continuously fed 10 [Mu]g F[B.sub.1]/g. These results establish that consumption of F[B.sub.1] causes dose-dependent and reversible elevations in the amounts of urinary sphingoid bases. The finding that 1 [Mu]g F[B.sub.1]/g (which does not, alone, alter urinary sphingoid bases) will sustain the elevation caused by previous exposure to 10 [Mu]g F[B.sub.1]/g raises the possibility that even low levels of fumonisins could be deleterious when an animal is occasionally exposed to higher amounts. KEY WORDS: Fumonisins; sphingolipids; urinary biomarker; rats

Sphingolipids are found in all eukaryotic and some prokaryotic organisms and participate in the regulation of cell growth, differentiation, and diverse cell functions including cell-cell communication, cell-substratum interactions and intracellular signal transduction. Nonetheless, the field of nutrition has given scant attention to these compounds so that little is known about the following fundamental questions: What is the fate of sphingolipids that are consumed in food? Does consumption of dietary sphingolipids affect the behavior of cells in the gastrointestinal tract or other organs? How do other factors in the diet affect sphingolipid metabolism? Several recent findings underscore the importance of these questions, for examples: 1) Sphingolipids are digested throughout the GI tract to ceramide and sphingosine, which are highly bioactive compounds that affect cellular regulatory pathways; 2) addition of sphingomyelin to a standard AIN diet (which is essentially devoid of sphingolipids) reduces the appearance of aberrant colonic crypts, and perhaps the number of tumors, in mice treated with a colon carcinogen; and 3) an enzyme of sphingolipid metabolism has been discovered to be the target of a class of toxic and carcinogenic mycotoxins called fumonisins. Given these recent findings, it is possible that some of the confusion that has arisen regarding the relationships between dietary fat and disease might be due to the lack of consideration of the sphingolipids that are also present. INDEXING KEY WORDS: sphingolipids; ceramide; mycotoxins; cancer

Sphingolipids are found in all eukaryotic organisms. However, little is known about the digestion, uptake and subsequent metabolism of these constituents of food. In this study, radiolabeled sphingolipids were placed in isolated intestinal segments of female CF1 mice, and the metabolism and distribution of the radiolabel were followed. Most of the sphingomyelin was degraded to ceramide and other products in all regions of the intestine, and increasing amounts of several (3H)-labeled sphingolipids appeared in the tissues. Small amounts of the radiolabel disappeared from the intestinal loops and appeared in liver within the first 30 to 60 min implying that neither intact sphingomyelin nor its metabolites are transported very efficiently from the intestine to other organs. There were different degrees of uptake and metabolism of sphingomyelin, (4,5-3H-sphinganyl)ceramide, and (3H)sphingosine. The (3H)sphingomyelin was also administered by gavage and the appearance along the intestine measured. After 90 min, 12% was found in the cecum and colon. These results establish that some of the sphingomyelin that enters the gastrointestinal tract is hydrolyzed and taken up by the intestine, with the lipid backbone being degraded or reutilized for complex sphingolipid synthesis; however, at least a portion passes into the large intestine. The appearance of bioactive compounds throughout the gastrointestinal tract may alter the behavior of intestinal cells.

Fumonisins are potent inhibitors of sphingolipid biosynthesis produced by several Fusarium species. Consumption of corn or corn products infected with F. moniliforme, or high levels of fumonisins, is associated with several animal diseases. In a 4-wk feeding study, the concentration of fumonisin B1 that caused nephrotoxicity in Sprague-Dawley rats was much less than that required to cause hepatotoxicity. This retrospective study shows a close correlation between the extent and severity of ultrastructural lesions and the degree of disruption of sphingolipid metabolism. The kidney was more sensitive to fumonisin B1-induced disruption of sphingolipid metabolism than liver with significant elevation of free sphingosine, free sphinganine, and the free sphinganine:free sphingosine ratio in rats fed 15, 50 and 150 microgams/g fumonisin B1. Accumulation of free sphinganine and elevation of the free sphinganine:free sphingosine ratio in urine closely reflected the changes that occurred in kidney. The accumulated sphinganine and elevation of the free sphinganine:free sphingosine ratio was associated with accumulation of cells in urine. Thus, urine rather than serum is the fluid of choice for detecting elevated free sphingoid bases generated as a consequence of fumonisin-induced kidney damage.

A study was done on the levels of sphingosine, sphinganine and complex sphingolipids in ponies given feed containing fumonisins and mycotoxins produced by Fusarium moniliforme. It was shown that consumption of feed contaminated with fumonisins alters sphingolipid profiles in serum by inhibiting sphinganine N-acyltransferase resulting to an elevation of serum sphingosine and sphinganine levels. This accumulation of free long-chain bases provide an early marker of exposure of animals to contaminated feed.

Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside [G.sub.D3] were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by [is greater than] 40% (P [is less than] 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P [is less than] 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 [micro]g of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or [G.sub.D3]) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis. J. Nutr. 130: 522-527, 2000. KEY WORDS: * proliferation * sphingomyelin * glycosphingolipids * colon cancer * mice