Toxicology Letters 140 /141 (2003) 459 /463 www.elsevier.

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Antinutritional effects of fumonisin B1 and pathophysiological consequences

M.R. Carratu ` a,*, T. Cassano a, A. Coluccia a, P. Borracci a, V. Cuomo b
a

Department of Pharmacology and Human Physiology, Medical School, University of Bari, Policlinico, Piazza G. Cesare, Bari 70124, Italy b Department of Pharmacology of Natural Substances and General Physiology, University of Rome, La Sapienza, P.le Aldo Moro 5, Rome 00185, Italy Received 15 September 2002; accepted 12 December 2002

Abstract Due to its structural similarity with sphingosine, fumonisin B1 (FB1) inhibits ceramide synthase (a key enzyme of sphingolipid biosynthesis) leading to an intracellular accumulation of sphingoid bases with a consequent increase of sphinganine/sphingosine (SA/SO) ratio. In adult male rats, dietary exposure to fumonisin induces a significant increase in both SA concentrations and SA/SO ratio in kidney, but not in liver and brain, as well as a significant reduction of body weight gain. Regarding the brain, the developing rat is more sensitive to FB1 than the adult rat. FB1 treatment produces in the forebrain and brainstem: (i) an increase in SA levels and SA/SO ratio, (ii) a reduction in myelin deposition, and (iii) an impairment of 2?,3?-cyclic nucleotide 3?-phosphohydrolase (CNP) activity. FB1 effects on myelin are similar to those produced by starvation (temporary removal of pups from dam during postnatal period), thus suggesting that hypomyelination could be due, at least partly, to a nutritional deficiency. Finally, FB1 reduces the uptake of folate in different cell lines. The resulting folate deficiency could explain the association of FB1 exposure with neural tube defects. # 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Fumonisin; Ceramide synthase; 2?,3?-Cyclic nucleotide 3?-phosphohydrolase; Folate uptake; Pathophysiology

1. Introduction Fumonisins are mycotoxins produced by Fusarium verticillioides ( /F. moniliforme ) and F. proliferatum which are found in corn crops worldwide

* Corresponding author. Tel.: /39-080-5478455; fax: /39080-5478444. E-mail address: mrc@farmacol.uniba.it (M.R. Carratu ` ).

(Bezuidenhout et al., 1988; Hopmans and Murphy, 1993). Ingestion of fumonisin B1, which contaminates food and feed (Shephard et al., 1990; Ueno et al., 1993), has been associated with leucoencephalomalacia in both horses (Marasas et al., 1988) and rabbits (Bucci et al., 1996), pulmonary oedema in pigs (Harrison et al., 1990), and nephrotoxicity and liver cancer in rats (Gelderblom et al., 1996). Although the effects of this mycotoxin on human are difficult to evaluate,

0378-4274/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(03)00042-0

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M.R. Carratu ` et al. / Toxicology Letters 140 /141 (2003) 459 /463

epidemiological studies show a high incidence of oesophageal cancer in certain areas of Transkei (South Africa) and also in China (Yang, 1980; Rheeder et al., 1992; Chu and Li, 1994). Due to its structural similarity with sphingosine (Bezuidenhout et al., 1988; Laurent et al., 1989), FB1 inhibits the ceramide synthase activity leading to an intracellular accumulation of sphingoid bases (mainly sphinganine relative to sphingosine), which mediate several key biological processes such as cell proliferation and DNA replication. Inhibition of ceramide synthase results in an increase of SA/SO ratio, which is considered a useful biomarker to assess fumonisin exposure. This ratio can be evaluated in tissues (such as kidney and liver) and biological fluids of both human and animals (Riley et al., 1993; Qui and Liu, 2001). Further mechanisms responsible for FB1 toxicity include inhibition of protein and DNA synthesis (Abado-Be cogne e et al., 1998) and lipid peroxidation (prevented by vitamin E), as shown in both primary rat hepatocytes (Abel and Gelderblom, 1998) and C6 glioma cells (Mobio et al., 2000). This paper briefly reviews the results of our recent studies as well as literature data dealing with FB1-induced disruption of sphingolipid metabolism and its consequences on folate deficiency and brain development.

Table 1 Body weight, feed consumption, and organ weight data of male Wistar rats fed control diet and fumonisin-contaminated diets for 1 week Parameters Initial body weight (g) Final body weight (g) Body weight gain (g) Feed consumption (g) Absolute liver weight (g) Relative liver weight (mg/g)a Absolute kidney weight (g) Relative kidney weight (mg/g)a Control 268.79/5.5 308.09/6.8 39.29/9.4 15.09/1.3 7.279/0.30 23.629/1.04 1.929/0.05 6.259/0.28 FB1 (4 ppm) 277.59/5.9 282.09/4.3 4.59/5.7* 15.69/1.6 9.459/0.45* 33.549/1.77* 2.089/0.12 7.49/0.53

a Organ weight (mg) to body weight (g) ratio. * P B/0.05 (one-way ANOVA; n /4).

contaminated diets produces a significant reduction of body weight gain and a significant increase of liver weight.

3. FB1 and nutritional deciency outcomes on developing brain As far as the brain is concerned, the developing rat is a more sensitive model to study FB1 effects than the adult rat since this mycotoxin is a hydrophilic, lipid-insoluble compound with a relatively large molecular weight. In this regard, it has been shown that FB1 administration from postnatal day (PND) 2 /12 produces the following effects in the forebrain and brainstem: (i) increase in SA levels and SA/SO ratio, (ii) reduction in myelin deposition, and (iii) impairment of 2?,3?cyclic nucleotide 3?-phosphohydrolase (CNP) activity (Kwon et al., 1997). Moreover, a significant decrease in body weight gain is observed in FB1treated animals. The effects produced by FB1 are similar to those induced by nutritional deficiencies. Temporary removal of pups from dam during postnatal period (6 /7 h per day from PND 3 to 12) significantly decreases body weight gain as well as myelin deposition and CNP activity in forebrain and brainstem. However, unlike FB1, nutritional deficiencies during postnatal period do not affect sphingoid base levels and SA/SO ratios in the brain. These data, summarized in Table 2, show that FB1 exposure affects sphingolipid metabolism

2. Effects of dietary fumonisin exposure on tissue levels of sphingoid bases In agreement with the literature data (Merrill et al., 1996; Riley et al., 1993), our recent studies (Solfrizzo et al., 2001) have shown that dietary exposure to fumonisin induces, in the adult rat, an increase in SA concentration and SA/SO ratio in kidney. In particular, mean kidney SA/SO ratios are found to be 5.8-fold higher in rats fed FB1contaminated diet than in rats fed control diet. Dietary exposure to fumonisin does not alter SA/ SO ratio and SA concentrations in liver and brain, thus suggesting that kidney could be considered the main target of this mycotoxin. Data relative to body weight gain, feed consumption, and organ weights are reported in Table 1. Exposure to FB1-

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Table 2 Effects of nutritional deciency (ND) and FB1 exposure in the developing rat Parameters Body weight gain Myelin deposition (forebrain and brainstem) CNP activity (forebrain and brainstem) SA concentration SA/SO ratio / /reduction; / /increase; NE /no effect. FB1 ND / / / / / / / / NE NE

in the central nervous system of developing rats by specifically impairing ceramide synthase activity as indicated by the increased SA levels. Furthermore, these findings point out that, unlike FB1, nutritional deficiency resulting in hypomyelination and impairment of CNP activity, does not alter sphingolipid biosynthesis since no change in brain SA levels are observed after starvation. Therefore, nutritional deficiency has no effect on ceramide synthase activity.

biosynthesis of these lipids (Stevens and Tang, 1997). In FB1-treated cells, the folate receptormediated transport of 5-methyltetrahydrofolate is inhibited in a concentration- and time-dependent manner. The sphingolipid levels of these cells also decrease in a concentration- and time-dependent manner, thus suggesting that the inhibition of 5methyltetrahydrofolate uptake in FB1-treated cells is mediated by changes in the sphingolipid composition. Moreover, a concurrent loss in the total amount of folate binding capacity in the cells is observed, as sphingolipids are depleted, thus suggesting a causal relationship between folate receptor density and vitamin uptake. Similarly, an inhibition of folate uptake is also observed in cultured primary embryonic cells treated with fumonisin. Collectively, these findings suggest that dietary exposure to FB1 could adversely affect folate uptake and potentially compromise cellular processes dependent on this vitamin. Finally, it should be pointed out that, since folate deficiency causes neural tube defects, some birth defects unexplained by other known risk factors may be caused by exposure to FB1.

4. Is there any link between FB1-induced sphingolipid depletion and folate deciency? 5. Conclusions Sphingolipids seem to play an important role in folate receptor function. This high-affinity receptor, a glycosylphosphatidylinositol (GPI)-anchored protein, is responsible for the transport of folate into cells of several tissues with elevated requirements for this vitamin. The folate vitamins play an essential role as cofactors in many biochemical reactions including the biosynthesis of purines and thymidine, the regeneration of methionine from homocysteine, and histidine metabolism. Cellular processes dependent upon folate can be compromised if dietary levels of this vitamin are insufficient or its transport into cells is affected. The GPI-anchored folate receptor is associated with membrane domains that are enriched in cholesterol and phospholipids. Depletion of cellular cholesterol has been shown to inhibit vitamin uptake by this receptor (Chang et al., 1992). Moreover, the importance of sphingolipids for folate receptor function has been demonstrated in CaCo-2 cells treated with FB1 that inhibits the Inhibition of sphingolipid biosynthesis, leading to an intracellular accumulation of sphingoid bases, still seems to be the main mechanism of fumonisin toxicity. The consequent increase in SA/ SO ratio provides a useful biomarker to assess exposure to this mycotoxin in both human and experimental animals. Investigations into the consequences of fetal exposure to this mycotoxin have also shown relevant developmental toxicity. The mouse fetuses surviving to birth have gross skeletal and tissutal abnormalities (Floss et al., 1994; Gross et al., 1994). Similarly to nutritional deficiency, FB1 exposure induces hypomyelination and it impairs CNP-specific activity. Factors affecting myelin deposition after FB1 exposure may include (i) antinutritional effects, (ii) consequences of increased brain SA levels, and/or (iii) SA-independent FB1-induced toxicity. Since postnatal nutritional deprivation also reduces myelin synth-

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M.R. Carratu ` et al. / Toxicology Letters 140 /141 (2003) 459 /463 MA104 cells inhibits receptor-mediated transport of folate. J. Cell Biol. 118, 63 /69. Chu, F.S., Li, G.Y., 1994. Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collect from the Peoples Republic of China in regions with high incidences of oesophageal cancer. Appl. Environ. Microbiol. 60, 845 /852. Dansky, L.V., Roseblatt, D.S., Andermann, E., 1992. Mechanisms of teratogenesis: folic acid and antiepileptic therapy. Neurology 42, 32 /42. Floss, J.L., Castell, S.W., Johnson, G.C., Rottinghaus, G.E., Krause, G.F., 1994. Development toxicity of fumonisin in Syrian hamsters. Mycopathologia 128, 33 /38. Gelderblom, W.C.A., Snyman, S.D., Abel, S., Lebepe-Mazur, S., Smuts, C.M., Van der Westhuizen, L., Marasas, W.F.O., Victor, T.C., Knasmu ller, S., Huber, W., 1996. Hepatotoxicity and carcinogenicity of the fumonisins in rats: a review regarding mechanistic implications for establishing risk in humans. Adv. Exp. Med. Biol. 392, 279 /296. Gross, S.M., Reddy, R.V., Rottinghaus, G.E., Johnson, G., Reddy, C.S., 1994. Developmental effects of fumonisin B1containing Fusarium moniliforme culture extract in CD1 mice. Mycopathologia 128, 111 /118. Harrison, L.R., Colvin, B.M., Greene, J.T., Newman, L.E., Cole, J.R., 1990. Pulmonary oedema and hydrothorax in swine produced by fumonisin B1, a toxic metabolite of Fusarium moniliforme . J. Vet. Diagn. Invest. 2, 217 /221. Hibbard, B.M., 1993. Folates and fetal development. Br. J. Obstet. Gynaecol. 100, 307 /309. Hopmans, E.C., Murphy, P.A., 1993. Detection of fumonisin B1, B2, and B3 and hydrolysed fumonisin B1 in corn containing foods. J. Agric. Food Chem. 41, 1655 /1658. Kwon, O.S., Schmued, L.C., Slikker, W., Jr., 1997. Fumonisin B1 in developing rats alters brain sphinganine levels and myelination. Neurotoxicology 18, 571 /580. Laurent, D., Platzer, N., Koh, F., Sauviat, M.P., Pellegrin, F., 1989. Macrofusin and micromonilin: two new mycotoxins isolated from corn infested by Fusarium moniliforme Sheld. Microbiol. Aliments Nutr. 7, 9 /16. Marasas, W.F.O., Kellerman, T.S., Gelderblom, W.C.A., Coetzer, J.A.W., Thiel, P.G., Van der Lugt, J.J., 1988. Leucoencephalomalacia in a horse induced by fumonisin B1 isolated from Fusarium moniliforme . Onderstepoort J. Vet. Res. 55, 197 /203. Merrill, A.H., Jr., Schmelz, E.M., Wang, E., Dillehay, D.L., Rice, L.G., Meredith, F., Riley, R.T., 1996. Importance of sphingolipids and inhibitors of sphingolipid metabolism as components of animal diets. J. Nutr. 127, 830 /833. Mobio, T.A., Anane, R., Baudrimont, I., Carratu , M.R., Shier, T.W., Dano, S.D., Ueno, Y., Creppy, E.E., 2000. Epigenetic properties of fumonisin B1: cell cycle arrest and DNA base modication in C6 glioma cells. Toxicol. Appl. Pharmacol. 164, 91 /96. Qui, M., Liu, X., 2001. Determination of sphinganine, sphingosine, and SA/SO ratio in urine of humans exposed to dietary fumonisin B1. Food Addit. Contam. 18, 263 /269.

esis (Wiggins et al., 1976), hypomyelination associated with FB1 treatment is supposed to be due, at least partly, to a nutritional deficiency. Finally, FB1-induced depletion of sphingolipids inhibits folate uptake leading to an intracellular deficiency in this vitamin. Folate deficiency during the first trimester of pregnancy is associated with an increased risk of neural tube defects (Hibbard, 1993; Butterworth, 1993; Dansky et al., 1992). Therefore, it is possible that some birth defects unexplained by the known risk factors might be linked to dietary FB1 exposure. For instance, high rates of neural tube defects have been observed among Hispanic (Canfield et al., 1996), for whom corn and corn products represent a sizeable portion of their diet. In conclusion, the experimental evidence that FB1-induced depletion of cellular sphingolipids inhibits folate uptake suggests that further investigations are needed to explore the possibility that this mycotoxin may contribute to some birth defects not accounted for by other known risk factors.

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