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As discussed earlier in section 1.5, Chapter One, there are over 20 diseases, such as Alzheimer’s, diabetes (type II), and Parkinson’s disease, that are associated with amyloidoses [35-38]. It has been suggested that amyloid fibril related diseases share common amyloid aggregate structures and pathological pathways regardless of the source of protein [39, 40]. Amyloid fibrils generated from non-amyloid proteins have also been reported to be toxic. For example, the amyloid aggregates of the SH3 domain from bovine phosphatidyl-inositol-3'- kinase and the amino-terminal domain of the E. coli hypF protein showed toxicity towards mouse embryo fibroblast cell line (NIH-3T3 cells) [41]. Bovine insulin has also been reported to form either toxic rigid fibrils with parallel β-sheet conformation or non-toxic filaments with anti-parallel β-sheet character under reducing conditions [42]. Therefore, it is important to study the biosafety of crystallin PNFs before exploring them for further applications.
Despite extensive studies in recent years [40, 43-47], the identity of the culprits of cytotoxicity associated with amyloidoses still remains unclear [48]. Although non-fibrillar oligomers are the main focus of attention, a significant number of studies have reported that mature amyloid fibrils can also produce a cytotoxic effect [49-51]. For toxicity studies (section 7.4), Hec-1a cells were cultured in Minimum Essential Medium Eagle (MEM) (section 7.4.1) and cell viability (section 7.4.2) was measured in the presence and absence of crude crystallin proteins, mature amyloid fibrils, and sonicated (fragmented) fibrils. These conditions were tested as fibril length may play an important role in cell toxicity [52, 53].
To cause the fragmentation of PNFs, sonication was investigated as it has previously been shown to cause the fragmentation of insulin amyloid fibrils [54, 55]. For fragmenting amyloid fibrils, mature crystallin PNFs were subjected to a range of sonication times (0-40 sec). The resulting solutions were assessed for fragmentation by TEM, and it was found that a sonication time of 20 sec was needed to fragment the crystallin PNFs (section 7.16.1). TEM images for PNFs, before and after sonication are shown in Figure 2.9.
Figure 2.9. Representative TEM images of crystallin PNFs, where (a) mature fibrils and (b) fragmented fibrils. Scale bar is 200 nm.
The crystal violet staining (CVS) assay (7.4.3), a method of similar accuracy as WST (water soluble tetrazolium salts) assays, [56, 57]was done to assess the potential toxicity of crystallin PNFs. The CVS assay is a simple and reproducible assay of cytotoxicity [58-60].In the CVS method, the principle involved to calculate the cell viability is based on the dye taken up by the viable cells in culture after the cells are stained with crystal violet (DNA of the cells is stained), and the resulting colour intensity is then measured spectrophotometrically at 570 nm.
The method of Gillies et al. (1986) [61]used to quantify the cell number in monolayer cultures as a function of the amount of the dye taken up by the cells has been extensively used with modifications for a wide number of applications including: to determine cytotoxicity or cell death produced by chemicals or toxins produced by microorganisms [62-64]and to determine cell proliferation [65] or cell viability [66]. Hec-1a cells were incubated with 10 mg/mL concentration of the crude protein, mature fibrils and sonicated fibrils, and the number of viable cells measured by the binding of crystal violet after 24 and 48 h. Even after 48 h incubation in comparison to the control, no substantial difference in the number of viable cells exposed to any of the given treatments was observed as demonstrated in Figure 2.10.
Figure 2.10. Hec 1a cell viability, in the presence of (a) mature fibrils, (b) fragmented fibrils, (c) crude crystallin protein (10 mg/mL), (d) control - nutrient medium + PBS, (e) 10% DMSO, and (f) no-cell sample. Cell viability is represented as % decrease/increase in number of cells as compared to control (d), nutrient medium is set to 100%. Error bars represent the standard deviation of the mean of three replicates. Blank columns 24 h and filled columns 48 h incubation time.
A further study was undertaken to assess whether there is any interaction between the cell and the proteins. The cells were pre-incubated in the absence of foetal bovine serum (FBS) to “starve” them overnight prior to the treatments (section 7.4.2). In comparison to the previous experiment there was an increase of cell viability in the presence of fibrils after 48 h incubation as depicted in Fig. 2.11. Similar results have been reported earlier for PNFs obtained from non- disease related proteins [67].
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The control (buffer + media) shows that the buffer conditions do not change the cell viability. 10% DMSO is toxic to cells and was therefore used as a positive control. The no-cell control also did not show any sign of viability, demonstrating that no contamination was present.
Figure 2.11. Hec 1a cell viability, in presence of (a) mature fibrils, (b) fragmented fibrils, (c) crude crystallin protein (10 mg/mL), (d) control - nutrient medium + PBS, (e) control - starvation medium + PBS, (f) 10% DMSO, and (g) no-cell sample. Cell viability is represented as % decrease/increase in number of cells as compared to control (d), nutrient medium is set to 100%. Error bars represent the standard deviation of the mean of three replicates. Blank columns 24 h and filled columns 48 h incubation time.
Our results suggest that Hec-1a cells are not adversely affected by the presence of crystallin amyloid fibrils at the studied concentration. There was also no indication that fragmented (sonicated) fibrils decreased cell viability, in contrast to literature reports [52].Instead, the in vitro studies suggest that the cells can perhaps utilise the fibrillar proteins as a source of nutrients, although the contribution of non-fibrillar protein components requires further investigation [68]. Previous studies have shown that the inherent stability of the β-sheet supramolecular structure adopted by the crystallins in the eye lens and the chaperone ability of α-crystallin must be crucial for preventing protein aggregation in vivo. Aoyama et al. (1993), have also shown that the presence of α-crystallins in cultured cells leads to an enhanced survival of these cells after a period of stress [69]. Thus, a further investigation is required to investigate that the increased cell viability is due to the maintained chaperone activity of crystallins in the fibrillar structure or due to the non-fibrillar crystallins present in the cell culture.
It is commonly agreed that the toxicity of fibrillar aggregates is caused by membrane disruption [70-73] and is often associated with Ca2+ release and oxidative damage [47]. Membrane disruption could be linked directly to the hydrophobicity and flexibility of the amyloid aggregate [47]. Therefore, the potential interaction of fibrils with cell membranes could differ [74], and further studies may be required using different cell lines with altered compositions of cellular membranes (see section 5.22, Chapter Five - attachment and proliferation of mouse fibroblast cells was investigated in the presence of PNFs).