RESULTS AND DISCUSSION - Cytotoxicity of selected Fusarium mycotoxins and sphingoid bases on

Cytotoxicity of selected Fusarium mycotoxins and sphingoid bases on the N2a mouse neuroblastoma cell line

3.3 RESULTS AND DISCUSSION

3.3.1 The MTT cytotoxicity assays

The MTT assays were performed to monitor cell viability by measuring the conversion of tetrazolium salts to yield formazan, the amount of which was considered proportional to the number of living cells.

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Figure 3.1: Dose response graphs of the effects of Fumonisin Bh Moniliformin and Fusaric acid on the N2a. cell line. p values <0.05 were taken as an indication of significant difference from the controls. p<O.05=·, p<0.01 = . ,

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Figure 3.2: Dose response graphs of the effects of sphinganine and sphingosine on the N2 a. cell line. p values

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Figure 3.3: Dose response graphs of the effects of Zearalenone, Deoxynivalenol and T -2 toxin on the N2a. cells.

p values <0.05 were taken as an indication of significant difference from the controls. p< 0.05=·, p<O.OI= ., P <0.001=· using Student's t-test are illustrated.

The results of the MTT cytotoxicity assays and the comparative effects of FBI, MON and F A on the N2a neuroblastoma cell line, are shown in Figure 3.1. The effects of the sphingoid bases Sa and So, as well as ZEA, DON, T2 on the same cell line are illustrated in Figures 3.2 and 3.3, respectively.

The exposure time of 48 hours was selected for the MTT assays on the basis of a dose response curve carried out over several time intervals. This helped determine a reasonable length oftime that N2a cells could be cultured in the microtitre plates with minimal levels of control cell death, taking into account the standard doubling rate of these cells in the volume of media per well. Support for selection of the 48 hour exposure time, comes from studies of Yoo et al. (1992) who showed that FBI inhibited cell proliferation (between 10 and 35IlM), and cytotoxicity (>35~M FBI) in LLC-PKI cells was preceded by a lag period of at least 24 hours during which cells appeared to be functioning normally. Although it is acknowledged that cytotoxicity is cell-type specific, in this study the 48-hour exposure period was considered appropriate for the MTT assays on the N2a neuroblastoma cell line.

To remove any bias in interpretation, all treated cells were assessed in parallel to their respective controls.

Most mycotoxins are difficult to dissolve in CCM, and in order to obtain a homogenous test solution, organic solvents are usually added to the media. Many studies have used FBI dissolved in methanol and/or acetonitrile: water (1: 1) for in vitro studies. However, based on the data of the United States National Toxicology Program (1999) that FBI is soluble in water to at least 20 mg.mr^l^,FBI was resuspended directly in the CCM in this study to ensure that all effects seen were the direct result of exposure to this mycotoxin, and not by exposure to the organic solvent.

Statistical analyses of the MTT bioassay results using Student's t-test indicate that there were significant differences between the overall effects of FBI (p=0.00095) in comparison to untreated N2a control cells across the concentration range (Figure 3.1; Table 3.1).

Statistically significant differences (p<0.01) were also noted specifically at 5, 50, 150, 200 and 250llM FBI in comparison to controls. The FBI trend line in Figure 3.1 indicates that there is a decrease in N2a cell viability with increasing FB I concentration (Figure 3.1).

Cell viability decreased from 75.7% at 5~M to 42.1% at 250llM FBI (Table 3.1). This decrease however was not strictly dose-dependent (Table 3.1). These results are supported

by studies by Norred et al. (1991), Shier et al. (1991) and Yoo et al. (1992) who have shown that FBI inhibits cell proliferation and is cytotoxic to many cell types. Inhibition of cell proliferation by FB I exposure usually occurs at concentrations that are lower than those which cause cytotoxicity.

Table 3.1:

Cone (J!M)

5 10 25 50 75 100 150 200 250

Mean percentage of N2a. cell viabilities (p value) following 48 hours exposure to fumonisin BI, fusaric acid and moniliformin in comparison to untreated N2a. control cells^t•

% Cell viability (p value)

FBI FA MON

75.7 (0.01) 108.4 (0.48) 106.9 (0.5)

36.7 (0.006) 88.9 (0.25) 83.9 (0.09)

85.6 (0.3) 134.1 (0.0009) 77.3 (0.13)

72.8 (0.028) 42.1 (0.006) 69.4 (0.04)

79.9 (0.06) 41.1 (0.004) 69.5 (0.02)

86.3 (0.11) 91.4 (0.3) 61.4 (0.002)

71.6 (0.003) 5l.6 (0.0004) 87.2 (0.14)

29.8(0.0001) 115.4(0.13) 68.7 (0.013)

42.1(0.0001) 33.2 (0.0003) 86.6 (0.08)

I Statistical analyses were done with comparison to matched controls. p values<0.05 were taken as an indication of significant difference from controls. p values < 0.05=·, p<O.O I = ., P <0.00 I • are indicated in Figure 3.1.

In an earlier study, Riboni et at. (1995) showed that FB I blocked So-induced differentiation of the N2a. neuroblastoma cell line. These authors established that conversion of So to CER was required and that de novo CER synthesis was important, as CER played a mediator role in the regulation of differentiation in these cultured cells. The structural similarity of the polyhydric alcohol moiety of FBI to So and Sa (Figures 2.4 and 2.5), allow FB I to be recognised as a substrate for CER synthase (Merrill et at., 1996b).

Merrill et al. (1993b) using microsomal preparations from cultured mouse cerebellar neurons showed that FBI was competitive with both sphingoid base and fatty acyl-CoA for CER synthase. Merrill and Sweeley (1996) further suggested that CER synthase recognized both the amino group (sphingoid binding domain) and the TCA side-chains (fatty acyl-CoA domain) of FBI. Spiegel and Merrill (996) also showed that inhibition of the enzyme induced a consequent intracellular accumulation of biologically active sphingoid bases and depletion of complex sphingolipids. These factors require consideration in the present study as compounding variables that may have initiated or exacerbated impairment of cellular transduction processes, cell differentiation, growth inhibition, cell mortality and/or mitogenicity of FBI on the N2a. cells.

In this study, -63% cytotoxicity was induced in the N2a cells at 10 IlM FBI (Figure 3.1), and at 200IlM, FBI induced -70% cell death following the 48 hour exposure period (Table 3.1). It is likely that the levels of cytotoxicity of FBI indicated by the MTT assay are related to the FB I-induced inhibition of So-induced differentiation of the N2a cells in culture. Tolleson et al. (1996b) found that the growth of keratinocytes and epithelial cells were inhibited by 42% when exposed to 1OJ.!M FBI. More recently, Mobio et al. (2000) reported that in the C6 glioma cells, FBI induced 10 ± 2% and 47 ± 4% cell death following 24-hour incubation period with 3~M and 541lM FBI respectively. In contrast, Galvano et al. (2002) exposed rat astrocytes to FBI, and found that FBI-treatment (48 hours, 72 hours and six days in vitro, at 10, 50 and 100llM FB 1) did not affect cell viability. These results highlight that the cytotoxic effects of FBI vary with cell type and duration of exposure and there are variable cellular responses.

Variable toxin susceptibility and the cytotoxic responses of cell types to FBI may be explained by the following. Although glial cells, astrocytes and neuroblasts are components of the CNS, the variability in cytotoxic response after exposure to FBI by the N2a neuroblastoma cdl line in comparison to glioma cells and astrocytes are potentially due to cellular, structural and biochemical differences. Reubel et al. (1987), Von Milczewskis (1987) and Holt et al. (1988) suggest that the varying cytotoxic responses of different cell types to particular mycotoxins may be due to the different metabolic activity of the target cells. The three most commonly encountered explanations for cell to cell variability in toxin sensitivity are differences in the amounts of target enzymes or other cellular functions which interacts with the toxin, the amounts of metabolic enzymes which activate a non-toxic precursor molecule into a metabolite with non-specific toxicity, and the efficiency of detoxification mechanisms that protects the cells and tissues that are less susceptible to toxic agent (Shier et al., 1991).

Thompson and Wannemacher (1984) suggest that the cytotoxic effects of a mycotoxin may also be influenced by the ability of a mycotoxin to bind to cellular receptors and/or penetrate cell membranes. This is dependent on the size, structural conformation of the toxin molecule, and its polarity. Furthermore, the relative significance of a molecular receptor of a mycotoxin is determined by its affinity to the active form of the toxin, its role in vital biochemical processes, the persistence of the lesion it bears, and the severity of the consequence of the lesion.

Inhibition of CER synthase by FBI causes the intracellular Sa levels to increase (Wang et al., 1991; Yoo et aI., 1992). The two most likely explanations for the increased cell death after inhibition of sphingolipid biosynthesis by fumonisins are that the accumulating free Sa (or a Sa degradation product) is growth inhibitory and cytotoxic to the cells (Stevens et aI., 1990; Hannun et aI., 1991; Sweeney et aI., 1996), and/or that more complex sphingolipids are required for cell survival and growth (Hanada et aI., 1992;

Radin, 1994; Nakamura ef aI., 1996). There is therefore the potential for misinterpretation of results of experiments using fumonisins as an inhibitor of CER biosynthesis, unless the effects of these sphingoid bases are also considered.

Ethanol was used as the solvent vehicle for Sa and So in combination with CCM in the MTT assays. Figures 3.2 and 3.3 show that although the viability of the N2a neuroblastoma cells decreased with exposure to the ethanol containing control serial dilutions, no significant differences were detected between any of the' test concentrations. This is indicated in Figure 3.2 by the control trend line, which indicates a gradual decrease in cell viability, with an increase in the percentage of ethanol in the CCM. To illustrate that the amount of ethanol used (100 ~l) to dissolve the mycotoxins did not induce an acute cytotoxic response in these cells, an ethanol-free control was run against the ethanol- containing CCM treated cells. The lack of a significant difference between the control and (control plus ethanol) treatment (p=0.99) indicate that the cytotoxicity induced by the ethanol solvent was negligible on this cell line.

Elevations of sphingoid bases disrupt normal regulatory mechanisms within cells, and are cytotoxic as indicated by exposure of the N2a cells to exogenous Sa and So (Figure 3.2).

The results of the MTT assay indicate that Sa and So were extremely cytotoxic at all the concentrations tested on the N2a cell line (Figure 3.2). Statistical analyses and comparison of So and Sa against their ethanol containing controls across the entire range, indicate statistically significant differences with p=1.1 x10-⁶ and p=1.9 x10-⁶ for So and Sa respectively. Comparison of the effects of Sa to So revealed that they did not differ significantly (p=O. 51). At each concentration tested however, statistically significant differences were noted (p<0.01) in comparison to the controls within the range. In addition, at all test concentrations cell viabilities were below 50% of the controls (Table 3.2).

Table 3.2: Mean percentage of N2a viabilities (p value) following 48 hours of exposure to sphinganine and sphingosine in comparison to ethanol treated N2a controls²•

Concentration (~M) % N2a. cell viability (p valuet

Sa So

5 32.3 (0.003 42.6 (0.0009)

10 38.7 (0.002) 36.2 (0.002)

25 27.4 (0.004) 30.7 (0.0012)

50 29.5 (0.0003) 29.3 (0.0007)

75 36.0 (0.001) 31.27 (0.0007)

100 45.8 (0.0l3) 40.72 (0.014)

150 34.8 (0.003) 29.26(0.0008)

200 49.1 (0.001) 46.45 (0.004)

250 45.5 (0.002) 40.22 (0.003)

2 Statistical analyses were done with comparison to controls run in parallel. p values <0.05 were taken as an indication of significant difference from controls. p values < 0.05=·, p<O.Ol = ., p <0.001· are indicated in Figure 3.2.

These results are supported by findings of previous studies on the N2a cell line by Hall et a!. (1988) and Riboni et al. (1995), who reported that addition of exogenous So rapidly elevated cellular CER levels in the N2a cells, suggesting that some effects of So may be mediated by its conversion to CER. The role of CER as an intracellular second messenger for TNF-a,

IL-P

and other cytokines; So, So-I-phosphate among other sphingolipid metabolites, has been demonstrated to modulate cellular calcium homeostasis, cell cycle progression and apoptosis (Yoo et al., 1992; Merrill et al., I993b). Elevated So and Sa in the N2a cells may therefore have acted via unscheduled initiation or inhibition of these pathways leading to disruption of metabolism of these endogenous molecules, leading to cytotoxicity and cell death. The maintenance of a low level of free sphingoid bases in cells is important because these compounds have considerable intrinsic biological activity and can be cytotoxic in high concentrations.

Another potential mechanism of action leading to decreased cell viability in the N2a cells exposed to Sa and So, may be via a mechanism similar to that proposed by van Echten et a!. (1990) and Mandon et a!. (1991). These authors showed that adding So to cultured mouse cerebellar neurons caused a decreased incorporation of [¹⁴C] serine into sphingolipids, apparently due to a down regulation of SPT in a time-and concentration dependent manner. A maximum decrease of 80% in this enzymes activity was achieved with doses as high as 50llM So homologues (Mandon et a!., 1991). At this concentration of Sa and So added to the N2a cell line in the present study on the N2a cells, approximately

70% cell cytotoxicity was induced following the 48-hour exposure period (Figure 3.2 and Table 3.2).

Chapter 8 in this dissertation documents the quantitative HPLC analyses of intra and extracellular levels of Sa and So in the N2a cells prior to and following exposure to FBI, and provides an indication of baseline levels of these sphingoid bases. In vitro and in vivo Sa and So levels are usually in the nanomolar range whereas in this MTT assay, they were tested at levels multifold higher; hence this level of cytotoxicity was not unexpected.

However, concentrations below 51lM Sa and So were not tested on the N2a cell line as this was a comparative study with the effects of the mycotoxins, and the dilution range to which the cells were exposed was standardised for both mycotoxins and sphingoid bases.

Future studies on these cells however will endeavour to establish the effects of exposure to exogenous Sa and So at concentrations below 5).lM.

Given that FBI inhibits CER synthase resulting in an increase in free Sa and So, and that the toxicity of FB I may be exacerbated via accumulation of these endogenous sphingoid bases, a point of interest in this study was that the equivalent amount of cell cytotoxicity was induced at 250llM FBI (Figure 3.1) and 5).lM So (~42%) (Figure 3.2). In addition, 32%

cell viability was noted with 51lM Sa. This is relevant, in that FBI may have induced inhibition of sphingolipid metabolism in these cells over the 48 hours leading to an increase in Sa and So, resulting in FBI initiating indirectly the equivalent levels of cell cytotoxicity. This hypothesis is supported by Gelderblom et al. (l995b; 1996b) where in fumonisin-treated primary rat hepatocytes, Sa: So ratios were maximally elevated at 111M FBI, whereas cytotoxicity was observed at >250).lM FBI.

Exposure to FA (Figure 2.11) at a concentration of 25 11M, induced proliferation of the N2a cells with viabilities of 134% being detected, i.e., viabilities 34% above controls (Figure 3.1). Fusaric acid may act on cytosolic copper ionlzinc ion (Cu2+/Zn2+) and/or mitochondrial manganese ion (Mn2+) - containing superoxide dismutase (SOD) enzymes that may be important for growth and survival of cancerous cells (Fernanadez-Pol et aI., 1982) or it may act by binding zinc which is a structural component of certain zinc finger DNA binding proteins that are involved in cell proliferation (Johnson and McKnight, 1989; Fernandez-Pol, 1991; Fernandez-Pol, 1992). Enhanced levels of formazan may therefore be interpreted as increased metabolic activity (Hanelt et aI., 1994)

and proliferation as observed with the N2a cells treated with FA. This hypothesis is supported by elevated cell viabilities of 108% and 134.1% detected at 5~M and 25~M FA in this study respectively (Table 3.1, Figure 3.1).

At higher concentrations of FA exposure (50,75, 150 and 250~M; p<O.OOl), higher levels of cell death were recorded, e.g., 41.1 % viability at 75 ~M FA and 33.2% viability at

250~M FA. However, using Student's t-test, when assessed across the entire range of concentrations, the effects ofF A on the N2a cells were not significantly different to that of control cells (p=O.l), or to cells treated with MON (p=0.9) (Figure 3.1).

Fernandez-Pol et at. (1993) reported similar findings with the human colon adenocarcinoma Lo Vo cells. In that study, within 24 hours of exposure to 500~M FA, most of the cells were blocked at random in the cell cycle and approximately 80% of the cells were dead after 30 hours. Vesonder et al. (1993) also found that FA was cytotoxic to the MDCK dog kidney fibroblast cell line, McCoy mouse fibroblast cells (MM), rat hepatoma tumour (RH) and CHO cell lines. Three human colorectal adenocarcinoma cell lines, SW 48, SW 480 and SW 742 were also sensitive to the inhibitory and cytotoxic effects of FA.

The cytotoxicity of F A may be attributed to the 5-butyt side chain (Figure 2.11), which because of its lipid solubility may allow better cellular penetrability of this mycotoxin. At

50~M and 200~M FA, 42.1% and 33.2% cell viability was detected in the N2a cell line respectively, following the 48hour exposure period. Telles-Pupulin et at. (1998) showed that FA affects mitochondrial energy metabolism by at least three modes of action, namely inhibition of succinate dehydrogenase, inhibition of oxidative phosphorylation, and inhibition of a-ketoglutarate dehydrogenase. The inhibition of oxidative phosphorylation seems to be the result of a direct action on the ATP synthase/adenosine triphosphatase (ATPase) without significant inhibition of the ATP/adenosine diphosphate (ADP) exchange. In isolated perfused rat liver, FA inhibits oxygen uptake and gluconeogeneis from pyruvate, the latter being strictly dependent on intra-mitochondrially generated ATP.

In the present study, cell viability is partially reflected by conversion of the MTT salt by succinate dehydrogenase enzymes of mitochondria of viable N2a cells. Lower cell viabilities may have been as a consequence of the inhibition of these enzymes by FA.

In this study, statistically significant differences were noted between MON-treated

(p=0.002) and control N2a cells across the range of the mycotoxin test concentrations. Cell

100

viabilities were between 61% and 69.5% with exposures between 50-200~M MON (Figure 3.1). Similar levels of cytotoxicity were noted in the RH, CHO and MDCK cell lines (Vesonder et aI., 1993).

Thiel (1978) reported that the principal mechanism of action of MON is believed to involve selective inhibition of mitochondrial pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase. Thiel (1978) reported that a 5 ~M concentration of MON caused a 50% inhibition of oxygen uptake by rat liver mitochondria with pyruvate as the substrate. Burka et at. (1982) showed that MON binds to PDH preventing entry of pyruvate into the TCA cycle, therefore decreasing mitochondrial respiration. Support for direct binding ofMON to PDH was also provided by Burka et at. (1982), who showed that rat brain transketolase and pyruvate dehydrogenase were inhibited by 59% by 1 OO~M

MON. More recently, decreased protein synthesis and induction of oxidative damage have

also been ascribed to MON (Norred et al., 1990).

The MTT assay uses reduced nicotinamide adenine dinucleotide (NADH) as a marker of cell viability. There are two main sources of NADH in cells catabolising carbohydrates;

namely glycolysis and the TCA cycle. The former is in the cytosol and the latter in mitochondria. Of these, the TCA cycle produces 10 times more NADH, so essentially the MTT assay measures this. If MON blocks the entry of pyruvate into the TCA cycle, it decreases mitochondrial respiration and therefore NADH production, and this would be reflected by decreased cell viabilities using the MTT assay. Following 48hour exposure to 50 ~ and 1 00 ~M MON, viabilities of 69.4% and 61.4% were noted for the mycotoxin treated N2a cells respectively.

In this study, sodium pyruvate supplementation of the CCM was essential to ensure N2a cell proliferation and survival; cells grown in CCM without sodium pyruvate ceased growing. This was considered significant in that it is possible that MON and the pyruvate supplement may have competed for the binding site on PDH. By adding high levels of MON as a suicide substrate of PDH, it may have potentially blocked or decreased mitochondrial respiration causing cell death. The data in the present study concur with earlier findings by Gathercole et al. (1986) who showed that pyruvate supplementation resulted in decreased MON-induced inhibition of PDH function. However in the present study, sodium pyruvate was added to the CCM for the N2 a cells at a concentration of

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Dalam dokumen The possible implication of selected Fusarium Mycotoxins in the aetiology of brain cancer. (Halaman 125-140)