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CHAPTER 2 Literature Review
2.15 FUMONISIN INDUCED DISRUPTION OF SPHINGOLIPID METABOLISM
2.15.1 Fumonisin-induced disruption of sphingolipid metabolism in cell systems
To fully understand the ways that disruption of sphingolipid metabolism could account for the cell damage caused by fumonisins, it is necessary to consider: -
• the multitude of functions of complex sphingolipids (Bell et aI., 1993),
• the potent bioactivity of Sa and its metabolites (Merrill et al.,1993a), and
• the parallel or branch metabolic pathways that can be affected by disruption of sphingolipid metabolism (Riley et aI., 1996; Merrill et aI., 1997).
Sphingolipids have been associated with many facets of cellular regulation (Merrill et aI., 1993a; Bell et al., 1993; Ballou et aI., 1996; Merrill et aI., 1997;
Kolesnick and Krenke, 1998; Perry and Hannun, 1998). Such associations could contribute to or modify the expression of fumonisin-associated diseases. Based on existing knowledge about some of the systems regulated by sphingolipids, mechanisms can be proposed whereby disruption of sphingolipid metabolism by fumonisins could account for the toxicity and carcinogenicity of these mycotoxins (Bose et aI., 1995; Strum et aI., 1995;
Witty et aI., 1996; Suzuki et aI., 1997).
Ceramide signalling is mediated by SM hydrolysis via enzymes that are not inhibited by fumonisin (Perry and Hannun, 1998). When fumonisins are added to cells for the purpose ' of inhibiting de novo CER generation, there is potential for accumulation of free sphingoid bases and their downstream sphingoid base-I-phosphates. Considerable data supports the hypothesis that fumonisin-induced disruption of sphingolipid metabolism is an important event in the cascade of events leading to cellular deregulation (Table 2.1). Several biochemical modes of action have been proposed to explain all, or some, of the fumonisin- induced animal diseases. Some invoke disruption of sphingolipid metabolism as the initial site of action, and there are also studies that hypothesize fumonisin-induced changes in key enzymes involved in cell cycle regulation, differentiation and/or apoptosis as initial or secondary sites of action (Merrill et al., 1995; 1996b; Riley et aI., 1996; Norred et aI., 1996;
Merrill et aI., 1997; Norred et aI., 1998; Wang et aI., 1999). Fumonisins may effect/affect carcinogenesis by altering sphingolipid metabolism. Cellular regulatory processes modulated by sphingolipids are known to be important in the control of normal cell growth, differentiation, apoptosis and immune response (Table 2.1). Fumonisins impact many of these processes and have a variety of effects depending on cell type and other factors.
The two most likely explanations for the increased cell death after inhibition of sphingolipid biosynthesis by fumonisins are that the accumulation free Sa (or a Sa degradation product) are growth inhibitory and cytotoxic for the cells (Stevens et aI., 1990;
Hannun et ai., 1991; Sweeney et aI., 1996), that complex sphingolipids are required for cell survival and growth, and loss of complex sphingolipid biosynthesis would be expected to alter cell behaviour, and lead to cell death. These hypotheses are based on findings with mutants of SPT, the initial enzyme of sphingolipid biosynthesis (Hanada et al., 1990; 1992;
Riley et al., 1999) and in studies with specific inhibitors of GSL biosynthesis (Radin, 1994;
Nakamura et al., 1996).
Table 2.1: Mechanisms for cell behaviour changes, growth inhibition and cytotoxicity induced by fumonisin via disruption of sphingolipid metabolism.
Repression of expression of PKC, stimulation of a cyelic adenosine Huang et al., 1995 monophosphate (AMP) response element in CV-I African green monkey
kidney cells (l-10flM FBI, 3 to 16 hours)
Decreased phorbol dibutyrate binding, increased cytosolic PKC activity, with Smith eta!., 1997 both exogenous Sa and FBI to J774A1 cells
Inhibition ofphorbol dibutyrate binding in short-term incubations using crude Yeung et al., 1996 cerebrocortical membrane preparation and both FBI and exogenous So
Activation of the mitogen-activated protein kinase (MAPK) in Swiss 3T3 Wattenberg et a!., 1996 cells with FB I
Over-expression of nuelear cyclin D 1 and increased cyelin-dependent kinase Ramljak et al., 2000 (CDK) 4 activity in rat livers obtained from a long-term feeding study and a
21-day feeding study with FB I
Dephosphorylation of the retinoblastoma protein, repression of CDK 2, and Ciacci-Zanella et al., induction of two CDK inhibitors in CV-1 cells with FBI 1998
Apoptosis inhibitor and protease inhibitor protection of CV-1 cells and Ciacci-Zanella and Jones,
primary human cells from FBI-induced apoptosis 1999
Increased TNF secretion in lipopolysaccharide-activated intra-peritoneal Dugyala et a!., 1998 macrophages from FBI-treated mice
Glutathione depletion and lipid peroxidation in cultured cells Kang and Alexander, 1996; Lim et al., 1996;
Abado-Becognee et a!., 1998; Abel and
Gelderblom, 1998; Sahu et al., 1998; Yin et al., 1998
2. 15.2 Mutagenicity of fumonisin Bl and related end-points
Fumonisin B1, FB2 and FB3 were shown to be non-mutagenic in the Salmonella assay against the tester strains TA 97a, TA 98, TA 100 and TA 102, either in presence or absence
I
of the S9 microsomal preparation at a range of concentrations from 0 to 10 mg/plate (Gelderblom and Snyman, 1991). The non-mutagenicity of FB J to Salmonella tester strain TAIOO at concentrations up to 100mg per plate was supported by Park et at. (1992).
Similar negative results were reported in gene mutation assays with FB J in Salmonella T A98 and TAl 00, as well as in SOS chromotest in Escherichia coli strain PQ37 (5, 16, 50, 166, 500~g/assay) in the presence and absence of metabolic activation, and differential DNA repair assays with E. coli K12 strains (343/753, uvrB/recA and 343/765, uvr+ rec+) (Knasmuller et at., 1997).
In contrast, Sun and Stahr (1993), using a commercial bioluminescent bacterial (Vibrio fischeri) genotoxicity test, claimed that FBI showed genotoxic activity without the metabolic activation of S9 fraction at a range of 5-20 ~g/m1. Fumonisin BJ and FB2 were non-genotoxic in an in vitro rat hepatocyte DNA repair assay at concentrations of 0.04- 80~Mlplate (and FB2 at 0.04-40~MI plate), as well as in vivo at 100 mg.kg-J
of FBI or FB2 (Gelderblom et al., 1989; 1992). The finding that FBI does not induce unscheduled DNA synthesis was confirmed in an in vitro assay in primary hepatocytes at concentrations from
0.5-200~M (Norred et at., 1990; 1992a; 1992b).
Gelderblom et al. (1988a; 1988b) found that fumonisins are complete carcinogens capable of inducing gamma-glutamyl-transpeptidase positive (GGTP) foci in the livers of both non- initiated and diethylnitrosamine (DEN) initiated rats. At low doses in rats, fumonisins appear to act primarily as tumour promoters (Gelderblom et at., 1995a) and induce increased apoptosis (Tolleson et al., 1996a). Single gavage doses of 50, 100 and 200 mg FBl/kg body weight significantly (p=0.05) inhibited hepatocyte proliferation in partially hepatectomized male Fischer rats (Gelderblom et al., 1995a). Inhibition of hepatocyte proliferation was also observed after dietary exposure to FBI (>50mg/kg diet) (Gelderblom et
at. ,
1996a). However, when evaluating the genotoxicity of several toxins to rat, FBI also inhibited DNA synthesis induced by epidermal growth factor (EGF) in primary rat hepatocytes (Gelderblom et at., 1995a; 1995b).Norred et
at.
(1992a) found FBI at 0.5-250 ~M, to have no effect on unscheduled DNA synthesis. Sheu etat.
(1996) on the basis of results in studies of FB I (1 0-1 OOO~g/ml) in BALB/3 T3 A31-1-1 mouse embryo cells concluded that FB I lacks in vitro transformingactivity. In these cells, FBI treatment produced transforming activity at 500 jlglml, but not at lower or higher concentrations.
Intraperitoneal injection of FBI induced an increased frequency of micronuclei in mouse bone marrow polychromatic erythrocytes at 25 and 100 mg.kg- I (Aranda et aI., 2000). Micronuclei form when chromosomal fragments behave independently of remaining chromosomes during the division of cells damaged by genotoxic agents, and the frequency of micronuclei is considered to reflect genotoxic damage to cells. It was concluded that FB I induces in vivo genotoxicity in the absence of in vitro mutagenicity in Salmonella (Aranda et aI., 2000). Sahu et al. (1998) showed that FBI induced DNA strand breaks in isolated rat liver nuclei.
On the basis of negative genotoxicity data from experiments covering several endpoints using the Ames test and in vitro and in vivo assays (Gelderblom et al., 1989; 1992; 1995a;
Norred et al., 1990; 1992a; Gelderblom and Snyman, 1991; Park et al. ,1992) and positive results in a non-validated type of bacteria test (Sun and Stahr, 1993), the overall conclusion is that there is inadequate evidence that FBI is genotoxic.
More recently however, Bever et al. (2000) reported that the incubation of methanolic extracts of Fusarium cultures with DNA in the presence of rat liver S9 proteins resulted in the formation of DN A adducts. The chromatographic characteristics of these unidentified DNA adducts suggest they are hydrophobic. Therefore, the possibility exists that compounds present in Fusarium fungi might alkylate DNA and participate in the induction of Fusarium induced tumours (Bever et aI., 2000; Howard et aI., 2001).