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CHAPTER 2 Literature Review
2.16 MOLECULAR MECHANISM OF ACTION OF FUMONISINS, SIGNAL TRANSDUCTION PATHWAYS, SPHINGOLIPIDS AND FUMONISIN Bl
Fumonisins cause compositional or oxidative damage to cellular lipids, which in turn cause molecular events culminating in oxidative damage to DNA and other critical macromolecules (Gelderblom et aI., 1996b; 1997; Abado-Becognee et aI., 1998; Abel and Gelderblom, 1998; Sahu et aI., 1998; Yin et aI., 1998). Wang et al. (1991) discovered that fumonisins inhibit CER synthase leading to disruption of de novo sphingolipid biosynthesis. The immediate consequences are accumulation of Sa and So, an increase in the Sa:So ratio, and depletion of complex sphingolipids. Recognizing the importance of sphingolipids in cell regulatory processes, including those related to proliferation and apoptosis, investigators proposed that the critical mechanistic step in fumonisin toxicity
was inhibition of CER synthase, starting a cascade of molecular events leading to cytotoxicity or neoplasia (Merrill, 1991; Riley et aI., 1994a; Hannun and Obeid, 1995;
Merrill et aI., 1997),.
Different hypotheses on the ability of FBI to alter signal transduction pathways and its potential role in carcinogenesis exist. Protein kinase C is a pivotal enzyme in cell regulation and signal transduction. Sphingosine is a potent and reversible inhibitor ofPKC.
Based on the ability of free sphingoid bases to inhibit PKC, it is possible that fumonisin repression of PKC activity is secondary to fumonisin-disruption of sphingolipid metabolism. Huang et al. (1995) treated green monkey kidney cells with FB I and found that fumonisin represses expression ofPKC and AP-1 dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element.
Since fumonisin did not alter protein kinase A (PKA) activity, it appears that cyclic AMP response element activation was independent of PKA. Huang et al. (1995) concluded that altered signal transduction pathways played a role in the carcinogenesis of fumonisins.
Similar results were found for baby hamster kidney cells treated with FBJ (Abeywickrama and Bean, 1992).
The activation of MAPK results in the modulation of transcription and activation of enzymes involved in signal transduction such as cytoplasmic phospholipase A2 (cPLA2), which releases arachidonic acid (AA) from membrane phopholipids. Arachidonic acid is the precursor of prostaglandins and leukotrienes, two inflammatory mediators that regulate gene expression and protein kinase activity among other effects. Fumonisin B I increases PKC translocation and stimulates MAPK. Pinelli et al. (1999) in an investigation on effects of FBI on the AA cascade in the W126 VA human bronchial epithelial cell line, found that FBI stimulated cPLA2 activity and increased AA release by a mechanism independent of PKC activation. The activation of cPLA2 was found to be a two-step process: the first being its phosphorylation by MAPK, and the second was a consequence of the increase in So inside and outside the cells after two hours, which is known to induce a rise in intracellular free calcium. Overall, this suggests that the effect of FBI on cells is partially dependent on the action of FBI on the enzymes involved in the cell cycle, such as MAPK and PKA, and on bioactive fatty acids, such as the prostaglandins and leukotrienes, and on disruption of sphingolipid metabolism (Pinelli et at., 1999).
In a study by Ramljak et al. (2000), which suggested the mechanism by which FBI acts as a carcinogen, the over-expression of cyclin Dl protein was shown in both pre-neoplastic and neoplastic liver specimens obtained from a long-term feeding study of FBI in rats. In rats fed FBI short-term, cyclin Dl protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in messenger ribonucleic acid (mRNA) levels of cyclin Dl. Two-dimensional electrophoresis of cyclin D 1 protein in FB I-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3 ~ (GSK-3~) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D 1. In FB dreated samples, GSK-3 ~ phosphorylated on serine 9 was detected. Activated protein kinase B (aPKB) appeared to be responsible for this inhibitory phosphorylation.
These findings suggest that over-expression of cyclin D 1 results from stabilization due to a lack of phosphorylation mediated by GSK-3~. An increase in CDK4 complexes with cyclin D 1 in FB I-treated samples was also observed. This study showed that the activation of aPKB leads to increased survival, inhibition of GSK-3~ activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle GIIS restriction point in hepatocytes (Ramljak et aI., 2000).
2.17 APOPTOSIS BY FUMONISlNS
2.17.1
In vivo
studies with rodentsFumonisin BI administered to different animal species increased apoptosis in vanous tissues (Lim et aI., 1996; Tolleson et aI., 1996a; Voss et aI., 1996a; Sharma et aI., 1997;
Bucci et aI., 1998; Haschek-Hock et aZ.; 1998; Ciacci-Zanella and Jones, 1999;
Lemmer et aI., 1999; United States National Toxicology Program, 1999). Increased apoptosis seems to playa role in toxic effects including tumour induction by FBI. In most studies, apoptosis is one of the observations on which the no-observed-adverse-effect level (NOAEL) is based. The dose level causing apoptosis depends on the duration of exposure, and can vary in rodents from 0.9 to 12mg FBdkg body weight (respectively in long-term and short-term experiments). Oxidative damage has also been indicated in the aetiology of toxic effects (Abel and Gelderblom, 1998).
Studies with rats have shown fumonisin-induced apoptosis in both liver and kidney. Male and female F344 rats were fed FBI (99, 163, 234, and 484 parts per million (ppm)) for 28 days and the organs examined histologically. Apoptosis was seen in hepatocytes at the lowest dose level, and the prevalence and severity increased with increasing dose. There was a dose dependent decrease in liver and kidney weights in the rats. The liver weight loss was accompanied by the induction of apoptosis and hepatocellular and bile duct hyperplasia in both sexes, with the female rats being more responsive at lower doses. The induction of tubular epithelial cell apoptosis was the primary response of the kidneys to dietary FB I. Apoptosis was present at all doses in the kidneys of male rats, and occurred in females only at 163, 234 and 484ppm FBI (Tolleson et aI., 1996a).
In a study on Fischer rats and B6C3F1 mice, FBI caused renal carcinomas in male rats and liver cancer in female mice. In male BD-IX rats, FBI caused hepatic toxicity and hepatocellular carcinomas. An early effect of FB I exposure in these target organs was apoptosis. However, there was also some evidence of oncotic necrosis following FBI administration, especially in the liver. Induction of apoptosis may be a consequence of CER synthase inhibition and disruption of sphingolipid metabolism by FBI. Dragan et al. (2001) hypothesise that FB I may be the first example of an apparently non-genotoxic agent producing tumours through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.
Sharma et al. (1997) administered subcutaneous injections (0 to 6.25 mg.kg-I
) of FBI daily for five days to male BALB/c mice. Their liver and kidneys were sampled one day after the last injection, and a decrease in kidney weight was observed. An evaluation of stained sections revealed dose-dependent FBI-associated hepatic and renal lesions in all groups.
Terminal uridine triphosphate (DTP) nick-end labelling (TUNEL) in liver and kidney sections confirmed the presence of dose-related apoptotic cells, at all treatment levels.
Thus, FBI produced apoptosis after a brief exposure to relatively low doses. The toxicity of FBI was greater than previously found in studies on oral toxicity (Sharma et aI., 1997).
2.17.2 Apoptosis in in vitro models
Increased apoptosis has been reported following in vitro exposure to FBI in turkey lymphocytes (Dombrink-Kurtzman et al., 1994), human keratinocytes, fibroblasts,
A
oesophageal cells and hepatoma cells (Tolleson et al., 1996b), and CV -1 monkey kidney cells (Wang et aI., 1996). Tolleson et al. (1996b) found that FBI inhibited incorporation of eH] thymidine by cultured neonatal human keratinocytes and HepG2 human hepatocarcinoma cells at 10-7 and 1O-4M, respectively. Fumonisin BI also inhibited clonal expansion of normal human keratinocytes and HET -1 A human oesophageal epithelial cells at 10-5M and growth in mass culture of normal human fibroblasts at 10-7M. The clonogenicity of normal human keratinocytes decreased to 45.5% of controls after exposure to 1 O-~ FB I for two days. However, no differences in the cell cycle distribution of cultured keratinocytes were noted after exposure to 1O-5M FBI for 40 hours. The viability of normal human keratinocytes and HET -1 A cells decreased as a result of FBI treatment, as determined by a fluorescein diacetate and propidium iodide (PI) flow cytometric cell viability assay. Fumonisin BI treated keratinocytes released nucleosomal DNA fragments into the medium 2-3 days after exposure to 1O-4M FBI and more DNA strand breaks were detected in attached keratinocytes exposed to 0-10-4M FB I using a terminal deoxynucleotidyl transferase-based immunochemical assay system. Furthermore, FB I-treated keratinocytes and HET -1 A cells developed morphological features consistent with apoptosis, shown by phase contrast microscopy, fluorescent microscopy of acridine orange (AcO) stained cells, and electron microscopy (EM) (Tolleson et aI., 1996b).
Tolleson et at. (1999) further examined the role of sphingolipid changes in FBI-stimulated apoptosis. Sphinganine accumulated rapidly, So levels remained unchanged, and CER decreased during FB I exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to FBI, Sa or N-acetyl So.
Co-exposure to N-acetyl So or ~-chloroalanine, which blocks Sa accumulation, partially protected cells from FB I-induced apoptosis. These results illustrate three sphingolipid- dependent mechanisms for inducing apoptosis; accumulation of excess CER, accumulation of excess Sa, and depletion of CER or complex sphingolipids derived from CER.
Incubation of human colonic HT29 cells, with FBI or the aminopentol (API) caused a significant reduction in cell number. The API was less potent with 50).lM API causing the same reduction in cell number (-30% after 24 hr) as 10).lM FBI. The reduction in cell number reflected increases in DNA fragmentation, and the percentage of apoptotic cells.
Both FBI and API caused the accumulation of Sa (25 and 35-fold by 10).lM FBI and 50).lM API, respectively); thus, concentrations of FBI and API that caused comparable reductions
in cell number were also similar with respect to elevation of Sa, which is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with myriocin (ISP-l) prevented the elevation in Sa, DNA fragmentation, and apoptosis induced by FBI.
Therefore, these effects of FBI on HT29 cells can be attributed to the accumulation of Sa (Schmelz et aI., 1998).
Wang et al. (1996) established that FBI-induced apoptosis, or cell cycle arrest, in CVl Mrican green monkey kidney fibroblasts, by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. The addition of FBI to CVl cells induced formation of DNA ladders, compaction of nuclear DNA, and appearance of apoptotic bodies. Fumonisin BI also induced cell cycle arrest in the G 1 phase in CVl cells (Wang et aI., 1996).
In a subsequent study, Jones et al. (2001) identified genes that inhibit FBI-induced apoptosis in CVl cells and two mouse embryo fibroblast (MEF) lines. A baculovirus gene p53, and inhibitor of apoptosis, CpIAP, protected these cells from apoptosis. CpIAP blocks apoptosis induced by the tumour necrosis factor (TNF) pathway, as well as via other mechanisms. Further support for the involvement of the TNF signal transduction pathway in FBI induced apoptosis, was the cleavage of caspase 8. Inhibition of caspases by the baculovirus gene p35 also inhibited FBI-induced apoptosis. The tumour suppressor gene p53 was not required for FBI induced apoptosis because p53-/- MEF undergoes apoptosis following FBI treatment. Furthermore, BcI-2 was not an effective inhibitor of FBI-induced apoptosis in CVl cells or p53+/+ MEF. These results provide information to understanding the mechanism by which FBI induces apoptosis (Jones et al., 2001).