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CHAPTER 2 Literature Review
2.26 EFFECTS OF FUMONISINS ON THE BRAIN AND BRAIN CELLS
2.26.2 Effects of fumonisins on myelin synthesis
2.26.4.4 Effects of fumonisin Bl on glycosphingolipids and gangliosides
Sialic-acid containing GSLs, gangliosides, are principal constituents of cell membranes and enriched in mature neurons. Sphingolipids are strong candidates as regulators of growth since they are enriched in neurons, and their levels and types, particularly gangliosides change significantly during development and differentiation. Furuya et al. (1995) observed an aberrant growth of dendrites of Purkinje cells depleted of membrane GSLs by FBI treatment. Fumonisin B I treatment resulted in less complex patterns of dendritic arborisation, suggesting a role of GSLs in neurite branching. Endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and SM is present abundantly in the somatodendritic region of these cells. Furuya et al. (1998) in a subsequent study investigated the effects of depletion of sphingolipids on the phenotypic
growth and survival of immature Purkinje cells and the ability of CER or other sphingoli pids to antagonize these effects. Inhibition of CER synthesis by ISP-1, decreased cellular levels of sphingolipids, decreased cell survival accompanied by an induction of apoptotic cell death, and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons (Furuya et at., 1998). These observations suggest that CER is a requisite for survival and dendritic differentiation ofPurkinje cells.
SHSY5Y lrk-A neuroblastoma cells are a cell clone transfected with NGF-receptor gene p140trk-A Addition of FBI (30-50~M) to SHSY5Ytrk-A human neuroblastoma cells resulted in a 40% decrease of the ganglioside content, and in a reduction of NGF-induced outgrowth of neuritic processes within four days. A comparable effect of FBI was found in a neuronal tissue culture system, resulting in a dose dependent inhibition of neurite outgrowth from embryonic chicken spinal cord explants. The effective concentrations were in the range of 20-50~M FBI. Similar concentrations have been reported to reduce axon elongation and branching of rat hippocampal neurons during a defined period of cell culture (Harel and Futerman, 1993; Schwarz et aI., 1995), as well as serum deprivation-induced neurite outgrowth of murine neuroblastoma cell lines.
Since FBI did not impair neurite growth, newly synthesised GSLs are not essential for axonal growth. This finding is in accordance with the study by Schwarz et at. (1995), in which incubation of cultured hippocampal neurons with FBI dramatically reduced the amount of cell surface gangliosides, as early as 24 hours after the initiation of treatment.
Schwarz et at. (1995) analysed the relationship between neuronal growth and sphingolipid metabolism by analysing the effect of FBI on neuronal development. Inhibition of GSL synthesis did not affect the formation of the parent axon during its emergence from the cell body. In hippocampal neurons, the most significant effect of inhibition of GSL synthesis was impairment of formation or stabilization of collateral axonal branches. Inhibition of sphingolipid synthesis results in a decrease in the number of axonal branch points/cell, whereas inhibition of sphingolipid degradation results in an increase in the number of axonal branch points (Schwarz et aI., 1995).
Schwarz and Futerman (1998) demonstrated that dendritic growth in hippocampal neurons also requires ongoing sphingolipid synthesis. With continuous incubation with FBI, dendritic growth rates were -25% slower than control cells, resulting in neurons with
shorter dendritic arbors and less dendritic branch points per cell, and readily apparent differences in morphology compared to control cells after 10-14 days in culture. In contrast, FBI had no effect on the initial growth of minor processes, which were destined to become dendrites, even in cells in which FBI affected the rate of axon growth. These data demonstrate that normal dendrite growth in hippocampal neurons requires sphingolipid biosynthesis, although the molecular requirements for sphingolipid synthesis may differ from those in axons.
de Chaves et at. (1997) presented evidence that FBI inhibits GSL synthesis in cultured primary rat sympathetic neurons by showing that FBI inhibited incorporation of CH] palmitate into several gangliosides in rat sympathetic neurons indicating that it inhibits GSL synthesis. The findings also indicate that newly synthesized GSLs are not essential for neurite growth, but that the lipid second messenger CER negatively regulates neurite growth.
2.26.4.5 Effect of fumonisin Bl on cell viability, protein synthesis and cell cycle
The involvement of lipid peroxidation on FBI induced cytotoxicity was investigated in the C6 glioma cell line to establish any relationship between cell viability, genotoxicity and cell division (Mobio et at., 2000). The study showed that FBI induced a reduction in cell viability, which was prevented by the antioxidant vitamin E, thus confirming the involvement of lipid peroxidation in the cytotoxicity induced by this toxin. Rat C6 glioma cell viability was not affected significantly up to 5-6J...1.M of FBI, then decreased to 60-65%
with 9-30 J...I.M FBI. Viability was never lower than 50% even with higher concentrations of 30-50 J...I.M FBI. The reduction in C6 glioma cells viability induced by FBI was prevented by vitamin E at 25J...1.M when cells were pre-treated before the addition of the toxin (p=0.01). Protein synthesis as determined by the incorporation of eH] leucine in C6 glioma cells was inhibited in a dose dependent manner by FBI after 24hour incubation. The inhibition was strong and linear (r=0.987) up to lO).lM. The IC50 concentration was 6J...1.M. For higher concentrations of FBI, the percentage of inhibition reached a plateau at about 75-80%. After 48hour incubation, FBI inhibited DNA synthesis exactly as after 24hour incubation.
A residu~l DNA synthesis was still going on. Protein synthesis was slightly less inhibited after 48 hours, as compared to percentage inhibition after 24 hours. This applied also to the C6 cells viability.
Using flow cytometry, the distribution of cells within the different cell cycle phases in control C6 cells was 36±2% for the Go/GI phase, 18.5±3% for S phase and 46±0.5% in G2iM phase When C6 glioma cells were incubated with FBI, the distribution of cells into the different cell cycle phases was modified. The number of cells in S phase decreased significantly, (p ::; 0.05), but not in a dose-dependent manner, from six up to 18~M FBI.
The number of cells in G2iM phase increased significantly (p ::; 0.05), but not in a dose-dependent manner from 3~ of FBI. The reason for this lack of dose dependency is not clear, however it is striking that low concentrations of FBI that are not highly cytotoxic are capable of inducing the cell cycle arrest. This effect could be closely related to genotoxicity or some epigenetic effects. This suggests that even low concentrations of FB I that do not affect cell viability are already capable of inducing cell cycle arrest in the G2iM phase (Mobio et at., 2000).
The synthesis of DNA as determined by CH] thymidine incorporation in C6 glioma cells was not significantly inhibited up to 5~M. With 10, 20 and 50~M of FBI, the inhibition of the DNA synthesis was of 27±4%, 37±4% and 27±0.5% respectively. Concerning the epigenetic effects, the results of Mobio et al. (2000) show that FB I induces significant hypermethylation in the DNA of C6 glioma cells in the concentration range of 9 to 18~M.
The lack of hypermethylation with FBI concentrations above 18~M could be related to an extreme cytotoxicity and also as a consequence of DNA damage. The extent of DNA damage induced by concentrations of FBI in the range of 6-18~M could favour the hypermethylation of DNA. In addition, methylated CpG sites can bind specific nuclear proteins such as MeCp 1 and MeCp2 (Nan et aI., 1993), which could conceivably block or modify the repair process (Tornaletti and Pfeifer, 1995).
The apparent discrepancy between the percentage of FBI-induced inhibition of DNA synthesis and protein synthesis in the C6 glioma cells, as related to the concentrations of the mycotoxin, could tentatively be explained by assuming that protein synthesis is the first target of FBI, thus confirming previous results (Abado-Becognee et aI., 1998). This could consequently decrease the intracellular level of DNA synthesis with the 48-hour incubation time. C6 cell viability was not modified while the protein synthesis inhibition was slightly lower. All these results reflect consequences of the residual DNA synthesis, and may allow cell multiplication to a new generation, which at its turn is being inhibited by FBI. Such a
pathway could generate mutated cell types due to possible DNA base modification induced by FB I in the previous cell generation.
2.26.4.6 Effects of fumonisin Bl on neuron extension and synapse formation
Neuronal growth is regulated by both extracellular and cellular determinants and proceeds by the addition of new membrane material at the growth cone. To determine whether lipid synthesis is necessary to maintain neuronal growth, Harel and Futerman (1993) examined the effect of FBI on the development of cultured hippocampal neurons. Ganglioside synthesis and content was reduced after FB I treatment, ganglioside GD 1 b was not detectable at the cell surface by immunofluorescence, and in fumonisin-treated cells no increase in axon length was observed. Addition of a fluorescent derivative of CER together with FBI reversed the effect, confirming that FBI acts via inhibition of CER synthase.
Normal axonal growth was restored by addition of exogenous CER derivative suggesting that depletion of GSLs directly caused the reduced axonal growth. The ability to restore normal function by CER replacement shows that these effects of fumonisins are due to loss of biosynthesis of a key sphingolipid (or sphingolipids). However, fumonisin induced disruption of sphingolipid metabolism causes numerous biochemical changes that could affect cellular regulation. It is difficult therefore to clearly demonstrate that a single biochemical perturbation could account for any specific effect on cell behaviour. This study showed that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons, inhibition of CER synthesis by FBI disrupted axonal outgrowth.
Using primary cultures of hippocampal neurons in a study on the regulatory roles for sphingolipids in the growth of polarised neurons, Futerman et al. (1998) found that sphingolipids play at least three distinct roles in regulating neuronal development. These are namely that CER enhances the formation of minor neuronal processes from lamellipodia, that glucosyiceramide synthesis is required for both normal and accelerated axon growth, and that at both of these stages of development, CER induces apoptotic cell death at high concentrations. These observations are consistent with the possibility that minor process formation and apoptosis are both regulated by CER-dependent signalling pathways, whereas axonal growth may require glucosylceramide synthesis to support an intracellular transport pathway (Futerman et aI., 1998).
Meivar-Levy and Futerman (1999) demonstrated that inhibition of CER synthesis by FBI for five days, resulted in up-regulation of the activities of three enzymes in the pathway of Gb3 synthesis; namely glucosylceramide, lactosylceramide and Gb3 synthases. Up regulation of the activities is due to an increase in maximal velocity (V max), with no change in Km values.
Aggregating brain cell cultures exposed to FB I (3-40 llM) for 10 days show decreases in both the total content of MBP and immunoreactivity of the oligodendrocyte membrane marker Gal C in the absence of general cytotoxicity (Monnet-Tschudi et aI., 1999).