.2 Morphological observation of Acanthamoeba sp. treated with Mn by light and scanning electron microscopy. To date, many techniques have been developed to determine the mode of cell death after exposure to xenobiotics and in this study, morphological observation due to effect of Mn on Acanthamoeba sp. was assessed by using light and scanning electron microscopy after incubation of the Acanthamoeba with Mn at their IC50 value. Fig. 2 shows a comparison between viable (Fig. 2a) and non-viable Mn-treated Acanthamoeba cells (Fig. 2b) are rounded and underwent apoptotic and necrotic type of cell death. Fig 2. Image of Acanthamoeba sp. under light microscope of untreated Acanthamoeba sp. (a) and Acanthamoeba sp. after treatment with IC50 value of Mn (b). Scanning electron micrographs of untreated (c) and Mn-treated Acanthamoeba sp. (d). Prominent acanthapodia can be seen in untreated Acanthamoeba whereas Mn- treated Acanthamoeba appear with severe damage on the cell surface with decreased number of acanthapodia structure. Based on the results obtained, untreated Acanthamoeba sp. shows the presence of acanthapodia with irregular shape of Acanthamoeba cells (Fig. 2a). Whereas the treated Acanthamoeba sp. shows the absence of acanthapodia with formation of vacuoles compared to the untreated Acanthamoeba cell (Fig. 2b). Mn- treated trophozoites of Acanthamoeba in the present studybecame rounded and floating in the culture medium perhaps as result of shortened or loss of acanthapodia (Fig. 2b). Contractile vacuoles were undetectable. The morphological change consequently inhibited the ability of the trophozoites to attach to the surface of the culture plate; in fact, the nearly spherical shape of trophozoite suggested the amoebae were beginning to become inactive and stressed out. Mn-treated Acanthamoeba became rounded in shape and underwent an encystment process due to unfavourable condition in the presence on Mn in its environment. To further evaluate the effects of Mn on the morphology of Acanthamoeba sp., the cells were viewed under scanning electron microscope. Fig. 2(c) shows untreated Acanthamoeba cell while Fig. 2(d) shows the Mn-treated cell. The untreated Acanthamoeba sp. showed the presence of acanthapodia, whereas the Mn-treated cell showed that the acanthapodia which is very important for adhesion to surface or substrate and for feeding [14], were almost absent. Collectively, the trophozoites became reduced in size, cystic cell appearance, and sunken food cups, loss of acanthapodia structure as well as wrinkle on the upper side of cell surface (Fig. 2d). In addition, thickened, broadened and elongated acanthapodia that are tightly attached to the surface of the substratum were also observed. This observation provides convincing evidence that Mn generate alterations of the Acanthamoeba sp. external morphology. Acanthameoba sp. turns to cyst stage when the cells are metabolically inactive as the environments are not conducive for them. Muller [15]) also stated that large numbers of vacuoles formed in early encystations as pinocytic activity are reduced. The treated Acanthamoeba sp. showed reduction in size with less acanthapodia present compared to the untreated cell. The external morphology also has been damaged. Amoebae that are having inactive throphozoites will later differentiate into cyst stage in which they are defending themselves from the unfavourable condition [7]. Siddiqui et al. [16] also stated that in cyst stage, the cell enclosed itself within a resistant shell and the trophozoites become metabolically inactive (minimal metabolic activity) after an initial burst of metabolic activity. Therefore, it can be concluded that Mn are toxic towards the Acanthamoeba cell. 3.3 Mode of cell death determination by fluorescence microscopy - Acridine orange/ propidium iodide (AOPI) staining. AOPI staining able to differentiate between viable and non-viable cell as well as the mode of cell death either apoptosis or necrosis. It wasemployed on Acanthamoeba cells after being incubated with Mn at its IC50 value. The stage of apoptosis either early or late apoptosis can be determined by the coloration of the cytoplasm. Green cytoplasm with orange stained nucleus indicates that the cell had undergone early apoptosis. Whereas for late apoptosis, both of the cytoplasm and the nucleus appear orange in colour as a results of the breakage of the nuclear envelop that led nuclear destruction [7]. Results obtained in this study shows that Mn played a role in inducing apoptosis or programmed cell death of Acanthamoeba sp. (Fig. 3). a b c d
19 Fatimah Hashim et al. / Procedia Environmental Sciences 30 ( 2015 ) 15 – 20 Fig 3. Green fluorescence of AOPI stained untreated Acanthamoeba sp. (a) and apoptotic and necrotic Acanthamoeba sp. after exposed to Mn (b). (v: vacuole, n: nucleus) (Mag : 400ൈ) Fig. 3 (a) shows cell with green cytoplasm with bright green fluorescent of intact nucleus and indicates that the cell were viable as the membrane-permeable AO dyes which are cationic are able to stain the negatively charged DNA in the nucleus and thus producing such colouration. Fig. 3(b) shows green colouration of cell with orange-stained nucleus to indicate the Acanthamoeba cell were non-viable. The Acanthamoeba cell that undergoes apoptosis are characterized by the green to orange of the cytoplasm with orange nucleus. PI dyes that are membrane impermeable are able to penetrate the cell and thus stain the nucleus with orange colour. This has indicated that the membrane of Acanthamoeba sp. has broken which allows the penetration on the PI dye. Nuclei of these cells are substantially fragmented and condensed to suggest the function of their membrane are disturbed even though the cells are coloured with green as in viable cells [7].