Microbiology (2004), 150, 33–43

DOI 10.1099/mic.0.26496-0

Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis
A. M. A. Nasirudeen, Yap Eu Hian, Mulkit Singh and Kevin S. W. Tan
Correspondence Kevin S. W. Tan mictank@nus.edu.sg

Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597

Received 16 May 2003 Revised 26 August 2003 Accepted 15 September 2003

Previous studies by the authors have shown that the protozoan parasite Blastocystis hominis succumbed to a cytotoxic monoclonal antibody with a number of cellular and biochemical features characteristic of apoptosis in higher eukaryotes. The present study reports that apoptosis-like features are also observed in growing cultures of axenic B. hominis upon exposure to metronidazole, a drug commonly used for the treatment of blastocystosis. Upon treatment with the drug, B. hominis cells displayed key morphological and biochemical features of programmed cell death (PCD), viz. nuclear condensation and nicked DNA in nucleus, reduced cytoplasmic volume, externalization of phosphatidylserine and maintenance of plasma membrane integrity with increasing permeability. This present study also supports the authors’ previously postulated novel function for the B. hominis central vacuole in PCD; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space. The implications and possible roles of PCD in B. hominis are discussed.

Programmed cell death (PCD) has been noted in all invertebrate and vertebrate multicellular organisms studied so far, including nematodes, insects, amphibians and mammals (Ellis et al., 1991; Raff, 1992; Vaux, 1993; Steller, 1995). It is thought to have evolved to regulate growth and development in multicellular organisms (Evan, 1994; Vaux et al., 1994) and as a defence mechanism against viral and bacterial infections (Shen & Shenk, 1995; Weinrauch & Zychlinsky, 1999). Unnecessary, damaged, infected and potentially harmful cells are deleted from surrounding healthy ones to ensure structural and functional homeostasis. Apoptosis, a form of PCD, is characterized by a unique pattern of morphological changes in both the cell nucleus and the cytoplasm. Although different cell types do not necessarily display all the hallmarks of apoptosis, characteristic features appear to be conserved in cells undergoing this form of cell death. These include shrinkage of the cell, preservation of membrane integrity with increasing permeability, chromatin condensation and DNA fragmentation, and externalization of plasma membrane phosphatidylserine (PS) residues (Saraste & Pulkki, 2000). Although it was long assumed that PCD evolved with multicellularity, several recent reports have suggested that similar cell-death machinery exists in protozoans such as Leishmania (Leishmania) amazonensis (Moreira et al., 1996),
Abbreviations: PCD, programmed cell death; PI, propidium iodide; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick-end labelling.

Trypanosoma cruzi (Ameisen et al., 1995), Trypanosoma brucei rhodesiense (Welburn et al., 1996), Dictyostelium discoideum (Cornillon et al., 1994), Plasmodium falciparum (Picot et al., 1997) and Plasmodium berghei (Al-Olayan et al., 2002). In our earlier study (Nasirudeen et al., 2001a), we showed that the human intestinal protozoan parasite Blastocystis hominis undergoes PCD when treated with a surfacereactive cytotoxic mAb (1D5), with typical features of apoptotic cells. Ageing agar cultures of B. hominis also displayed ultrastructural features of apoptosis (Tan et al., 2001). The presence of an apoptotic response to undesirable external stimuli led us to investigate if a similar phenotype is observed upon drug exposure. Drugs such as staurosporine have been shown to cause cell death in mammalian cell lines (Nakazono-Kusaba et al., 2002; Koh et al., 1995). In protozoan parasites such as P. falciparum, chloroquine has been reported to induce cell death (Picot et al., 1997). T. brucei rhodesiense undergoes apoptosis when treated with concanavalin A (Welburn et al., 1996). Free-living unicellular organisms have also been reported to undergo apoptosis. Davis et al. (1992) observed apoptoticlike nuclear degeneration in conjugating Tetrahymena cells while Ludovico et al. (2001a, b) reported that PCD can be induced by acetic acid in Saccharomyces cerevisiae. Taken together, these observations suggest that many unicellular organisms have the unusual capacity to activate a complex cell-death programme similar to their multicellular counterparts. This feature has been postulated to aid these microorganisms in regulation of growth and developmental

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The cells were labelled by adding 50 ml TUNEL mix followed by incubation for 60 min at 37 uC in a dark. Stained parasites that fell within the M1 region were represented as a percentage of total cells analysed. hominis after exposure to 561027 M metronidazole. included in the kit. hominis isolate B was obtained from a local patient and maintained axenically in Iscove’s modified Dulbecco’s medium (IMDM) containing 10 % horse serum (Ho et al. Nick-labelling of internucleosomal DNA fragments. Cells were washed twice in 0?1 M sodium cacodylate buffer with 5 % sucrose and fixed sequentially in each of the following fixatives: 1 % glutaraldehyde in 0?5 M sodium cacodylate buffer pH 7?2 for 3 h. 1993). Green fluorescence was detected using a 525 nm band-pass filter. appeared smaller and darker (Fig. Metronidazole. Metronidazole is a nitroimidazole antibiotic used clinically to treat B. Metro- nidazole (561027 M) was used to induce cell death in B. Stock solutions of metronidazole M-1547 (Sigma) were prepared in double distilled water and further diluted in IMDM to obtain the desired concentration. 1989). we have employed flow cytometry and electron microscopy to study the effects of metronidazole on B. followed by two washes in buffer. Cell shrinkage was confirmed using flow Microbiology 150 of PS was detected using an Annexin V/FITC kit (BenderMed Systems) as described previously (Nasirudeen et al. Exclusion of PI from these cells was taken as a quantitative marker for membrane integrity (Cornillon et al.. hominis and arrived at an ID50 value of 3?3461027 M for metronidazole.. and may also provide us with clues on host– parasite interactions and pathogenesis (Knight. 2002).. Necrosis was induced by heating the cells in an 80 uC water bath for 15 min. M. 1b). hominis infections as well as for the treatment of anaerobic infections in humans (Garavelli. Briefly. 2001a). Ok et al. 9 and 12 h. Cells were inoculated into pre-reduced IMDM to give a final concentration of 26106 cells ml21. hominis. 1997. Two controls were included in the experiments: (i) cells grown in normal culture conditions (IMDM with 10 % horse serum). abdominal pain.7 software program. After two washes with PBS. Metronidazoletreated and untreated cells (26106 cells ml21) were washed twice with 1 ml PBS and fixed with 4 % formaldehyde/PBS for 25 min at 4 uC. DNA was electrophoresed in 2 % agarose gels at 100 V for 2 h and visualized by using an UV transilluminator. (ii) necrotic cell control of B. Llibre et al. The samples were measured by flow cytometry (Coulter Epics Elite ESP) for FITC/PI fluorescence and results were displayed using the WINMDI 2. 190 ml calcium-containing binding buffer. A region (M1) was defined to exclude background fluorescence by unstained cells. The cells were then processed according to the procedure used by Moe et al. hominis is a controversial issue: some authors consider it to be a pathogen (Lee. Metronidazoletreated B. Cells were harvested at 3. 9 and 12 h intervals. Membrane integrity analysis and detection of PS externalization. hominis. (1999) and examined using a Philips EM280S electron microscope. Treatment with metronidazole to induce cell death. In the present study. Metronidazole. 1994). Dunn & Boreham (1991) developed an assay to measure the sensitivity of drugs against B. 16107 cells were incubated and harvested at 3. 34 . Nasirudeen and others 0?21 mg ml21 FITC–Annexin V and 2?5 mg ml21 propidium iodide (PI) were added sequentially. In the presence is a 5-nitroimidazole drug used in the treatment of blastocystosis and other anaerobic infections.. hominis infections. Reproducibility of results. The pathogenicity of B. 9 and 12 h intervals. Eighty microlitres of equilibration buffer were added and the cells were incubated at room temperature for 5 min. 1990. RESULTS Cell shrinkage in metronidazole-treated cells Phase-contrast microscopy revealed that B.. Transmission electron microscopy analysis. DNA ladder assay.. Similar conditions were followed for ultrastructural studies except that necrosis was induced by heating the cells in an 80 uC water bath for 15 min. 2-methyl-5-nitroimidazole-1-ethanol. The samples were washed with PBS and the pellet was resuspended in 250 ml PBS prior to flow cytometry using an argon-ion laser tuned to 488 nm. Symptoms reported in B. 26106 cells incubated in the presence and absence of metronidazole were harvested at 3. 6. deoxynucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick-end labelling (TUNEL) was performed using the ApoAlertTM DNA Fragmentation Assay kit (Clontech). 1991. 1989). Terminal METHODS Parasite and growth conditions. clear central vacuole and peripheral cytoplasm (Fig. The detection of loss of membrane permeability and exposure and absence of metronidazole. 1999). Hager & Rapp. humidified incubator.. B.A. were used as a positive control. All experiments were repeated at least twice. Metronidazole was then added to a final concentration of 561027 M. In the presence and absence of metronidazole. Although metronidazole has been used clinically as a drug to treat B. 6. 2 % osmium tetroxide in 0?1 % potassium ferrocyanide solution for 1 h and then two washes in 0?5 M cacodylate buffer. Apoptotic U937 cells (histiocytic lymphoma cells). Cells were grown anaerobically (Concept Plus Anaerobic Workstation. whereas others conclude that it is a harmless commensal (Rosenblatt. Several reports show the parasite to be more pathogenic in immunosuppressed patients such as those with AIDS (Llibre et al. 1a). 9 and 12 h for flow cytometry analysis and phase-contrast microscopy. 1999). 6. One millilitre of 20 mM EDTA was then added to terminate the tailing reaction. which was performed once. hominis infection include diarrhoea. little effort has been made to evaluate the pattern of death induced by this drug. the gels were photographed with a Polaroid camera. Cells were washed twice in 1 ml PBS (pH 7?4). processes. flatulence. hominis cells grown in normal culture conditions displayed healthy morphology. Sun et al. 1989. except for transmission electron microscopy analysis. Ruskinn Technology) at 37 uC. as indicated by a spherical shape. hominis cells. The present study demonstrates characteristic features of PCD in the human intestinal protozoan parasite B. hominis was obtained by incubating cells with 0?1 % sodium azide and harvesting 24 h post-treatment. the pellet was resuspended in 5 ml permeabilization solution (0?2 % Triton X-100 in PBS) and incubated on ice for 5 min. 6.. Then. A. 1991. 56106 cells ml21 were incubated and harvested at 3. however. 1992). An apoptotic DNA ladder kit (Boehringer Mannheim) was used to extract DNA from apoptosis-induced and uninduced cells according to the manufacturer’s instructions. nausea and constipation (Ok et al.

Note cell shrinkage. (c) Flow cytometry analysis of cell size and granularity. (a) Cells grown in normal culture conditions. green) is due to dead cells and debris while the population on the right (R1.Metronidazole-induced apoptosis in Blastocystis Fig. 20 mm.org 35 . The population on the left (R2. (b) cells exposed to 5610”7 M metronidazole.sgmjournals. Bars. red) could be a mixture of healthy cells and cells undergoing PCD. http://mic. Dot plots show two distinct populations of cells. hominis cell size as determined by light microscopy and flow cytometry. condensed cytoplasm and darkening of cells after exposure to metronidazole. 1. Effect of metronidazole on B.

giving red fluorescence. Increase in PS translocation was detected by flow cytometry in metronidazole-treated cells over a period of 9 h. this is followed by the incorporation of biotinylated deoxyuridine triphosphate by terminal deoxynucleotidyl transferase into the sites of DNA breaks. Annexin V (a PS-binding protein) preferentially binds PS in a calcium-dependent manner. The results were analysed by flow cytometry (Fig. 1998) while maintaining membrane integrity. untreated cells. the Annexin V binding of metronidazoletreated cells dropped to 30?78 %. respectively. Metronidazoletreated cells. Results are based on two replicates and are shown ±SD. Note gradual increase in PI uptake in metronidazoletreated cells. hominis cells DNA fragmentation and the display of DNA ladders. TUNEL relies on the specific binding of terminal deoxynucleotidyl transferase to exposed 39-OH ends of DNA followed by the synthesis of a labelled polydeoxynucleotide molecule. Absence of DNA laddering in metronidazoletreated B.A. A. M. a population of cells with dramatically increased Annexin V binding began to emerge in these cultures and increased rapidly thereafter. Necrotic cells showed only 9?90 % Annexin V binding. Approximately 30 % of the B. Cells grown in normal culture media showed 1?45 % PIpositive cells at 3 h and 1?13 % PI-positive cells at 12 h. Nuclear DNA is first exposed by proteolytic treatment. hominis dipped to 34?8 %. Agarose gel electrophoresis of B. Presence of in situ DNA fragmentation in the nucleus of metronidazole-treated cells Endonuclease activity was evaluated with the TUNEL assay.. A positive control consisting of apoptotic U937 cells (Boehringer Mannheim) showed a DNA-laddering profile typical of apoptosis (Fig. Nasirudeen and others cytometry where metronidazole-treated cells showed progressive reduction in cell size (Fig. B. before treatment with metronidazole. this asymmetry collapses and PS is exposed onto the outer surface of cells (Gatti et al. In normal cells. The signal is amplified by avidin–peroxidase. Control cells in normal growth media showed minimal DNA fragmentation. Such activity in parasites cultured in normal growth media was approximately 2?0 and 3?1 % at 3 and 9 h. At 12 h. This dye was used together with Annexin V. 1c). hominis cells were PI-positive when treated with 0?1 % sodium azide for 24 h. often of 200 bp and multiples thereof. 4. 5). hominis cells undergoing PCD. while the simultaneous presence and absence of PI accumulation indicates apoptotic or necrotic states. 2. 36 . An Annexin V assay was used to detect the presence of PS on the plasma membrane. No DNA ladder was observed in any of the experiments. hominis DNA in the presence and absence of metronidazole and in the presence of 0?1 % sodium azide is shown in Fig. PI can only enter cells with an altered plasma membrane and then stain the nucleic acid. in agarose gel electrophoresis are common features of apoptosis. hominis cells that were exposed to 0?1 % sodium azide (necrotic control) showed 11 % TUNEL (Fig. TUNEL of metronidazole-treated B. Microbiology 150 Fig. only 2?57 % of the cells were Annexin V-positive. A short incubation with PI was used to investigate membrane integrity in B. Dot plot displayed two distinct populations of cells. under physiological conditions. Flow cytometry data revealed a gradual increase in dying cells (increase in cell permeability) due to metronidazole treatment. hominis cells by flow cytometry. Annexin V binding in these cells increased from 32?39 % at 3 h to 51?09 % at 9 h as compared to 2?72 % of control cells at 9 h. Approximately 12?4 % of metronidazole-treated cells were TUNEL labelled by 3 h and 58?8 % were maximally labelled by 9 h. At 12 h. 3. 2): 2?87 % of the metronidazole-treated cells were PI-positive at 3 h and 4?60 % were PI-positive at 12 h. X. Preservation of membrane integrity and externalization of PS in cells undergoing PCD Loss of cell viability is most often measured as loss of membrane integrity. Necrosis was achieved by treating cells with 0?1 % sodium azide for 24 h and the PI-positive value is represented as a horizontal line for comparison. As shown in Fig. Membrane integrity assay of B. and cellular debris. enabling conventional histochemical identification of PCD by flow cytometry. within 3 h of metronidazole treatment. Formation of DNA strand breaks as a consequence of endonuclease activity can be detected by the TUNEL assay and expressed as a percentage of TUNEL-positive cells. Exclusion of PI by membrane-intact cells was scored as preservation of membrane integrity. which binds PS exposed during apoptosis. The population on the right (R1-gated) represents larger cells (healthy and early apoptotic cells) while the population on the left (R2-gated) represents late apoptotic and necrotic cells. However. respectively. &. 4). PS is segregated to the inner leaflet of the plasma membrane. During the early stages of apoptosis/PCD.

1000 DISCUSSION 500 Fig.sgmjournals. Metronidazole enters cells and mitochondria by simple diffusion. Representative dot plots and graphical representation of similar experiments showing externalization of PS in B. hominis impaired its growth at an ID50 value of 3?3461027 M.Metronidazole-induced apoptosis in Blastocystis Fig. Metronidazole-treated cells show a significantly higher percentage of Annexin V-labelled cells relative to the control cells. Cells that underwent necrosis presented a morphology distinctively different from that of apoptotic B. was visible within the nucleus (Fig. which is usually seen as a crescentic mass at the nuclear periphery. 9 and 12 h metronidazole treatment. the nuclei appear distinctively smaller and the nuclear chromatin had segregated as distinct clumps along the nuclear periphery (Fig. appearing empty or containing some inclusions. Ultrastructural features of apoptosis in metronidazole-treated cells Transmission electron microscopy micrographs showed that cultures in normal growth media had classical B. Agarose gel electrophoresis of B. In the lower graph. results are given based on two replicates and are shown ±SD. 9 and 12 h. 100 bp marker. 2. 6. The presence of large cytoplasmic vacuoles. 4. suggesting that most or all of the apoptotic bodies had been released (Fig. 6b). 6a). 3. 6a). 6c) and the presence of membrane-bound electron-dense particles (apoptotic bodies) in the central vacuole (Fig. respectively. a number of cells showed a break in the central vacuole. Note the absence of a DNA ladder in lanes 3–12. 6d) were noted. control untreated parasites 0. 3. hominis. hominis. Dunn & Boreham (1991) showed that metronidazole treatment of B. hominis morphology (Fig. http://mic. B. 3. 12. hominis cells treated with metronidazole showed 1 bp 2 3 4 5 6 7 8 9 10 11 12 morphological changes suggestive of apoptosis. At 12 h. apoptotic U937 cells. In mitochondria. 6e). 6. Normal DNA chromatin. the invagination of the cytoplasm into the central vacuole (Fig. 6f). In the presence of metronidazole. 37 . 3–7. metronidazole competes efficiently with the natural electron acceptor for electrons. hominis cells grown in normal culture conditions (&) and those exposed to metronidazole (X). 0?1 % sodium azide treatment. 8–11. and the central vacuole seemed devoid of any electrondense particles (Fig. The apoptotic bodies in the central vacuole appeared to be released into the extracellular space. Lanes: 1. Necrotic cells were electron-lucent and showed swelling of organelles such as mitochondria and nucleus. In contrast. hominis DNA. respectively. We therefore reasoned that the concentration of 561027 M used in this study should be sufficient to induce cell death in a majority of the parasites in culture.org Metronidazole is an antibiotic used for the treatment of anaerobic microbes including B.

hominis cell displaying normal morphology and DNA chromatin seen as a crescentic mass (arrow). In cells grown in normal media. 2001a). Note significant TUNEL intensity increase in metronidazole-treated cells. hominis cells (<10 % of cells PI-positive). This could be due to loss of cytoplasmic fluids and denaturation of proteins in apoptotic cells (Huppertz et al. (c) Large cytoplasmic vacuoles (V). cell shrinkage. there was no significant increase in the uptake of PI. cytoplasm pinching inwards into central vacuole (arrow). hominis. Flow cytometry analysis revealed that metronidazoletreated B. (b) Segregation of condensed chromatin to the nuclear periphery and condensation of nucleus (arrow). (d) Membrane-bound organelle-containing vesicles within the central vacuole. &. Note clear central vacuole in necrotic cells. 1999). electron microscopy is often employed to observe key ultrastructural features. In apoptosis research. Fig. deposition of membrane-bound apoptotic bodies. results are given based on three replicates and are shown ±SD. When treated with 561027 M metronidazole. Nasirudeen and others Fig. maintenance of cytosolic organelle structure and size (unlike necrosis where the organelles swell and appear disoriented) and heavy vacuolization clearly provide ultrastructural evidence of apoptosis in metronidazole-treated B. only a small percentage of cells became permeable to PI while most of the cells maintained membrane integrity. X. These were much lower than those observed in control necrotic cells. (f) Necrotic cells with swollen mitochondria and nucleus and chromatin seen in large clumps in necrotic cells. Flow cytometry data further supported cell shrinkage observed under light microscopy. A. mitochondria. hominis clearly showed ultrastructural characteristics of apoptosis. CV. Transmission electron micrographs of B. The reduction of the metronidazole nitro group results in the synthesis of cytotoxic radicals (R-NO2) (Kulda. Transmission electron micrographs of metronidazole-treated B. Upon exposure to metronidazole. hominis. hominis preserved its membrane integrity with PI-exclusion levels comparable to those of normal B. The present study shows that metronidazole causes apoptotic-like death in B. the antimicrobial effect of 2 metronidazole depends on its metabolic reduction within the target cell resulting in the release of cytotoxic radicals (Kulda. 1 mm. 1999). central vacuole. M. Hence.. (a) Healthy B. Organisms susceptible to 5-nitroimidazoles transfer electrons generated by their electron-transport systems to the nitro group of the drugs and not to their natural electron acceptor (Kulda. 6. nucleus. vacuole. hominis cells exposed to metronidazole for 3–12 h. resulting in a large intramembranous space. light microscopic data showed cell shrinkage and darkening of the cytoplasm in B. we described similar apoptotic-like features in this organism in response to a cytotoxic antibody exposure (Nasirudeen et al.A. Metronidazole-treated cells. 38 Microbiology 150 . the cells have apparently released most or all of their apoptotic bodies. V. hominis cells by flow cytometry. 1999). N. untreated cells. indicating normal growth and an intact plasma membrane. Representative histograms and graph showing in situ DNA fragmentation analysis (TUNEL) of B. In the lower graph.. 5. M. Nuclear condensation. 1999). In an earlier study. Bars. which gave PI levels of approximately 30 %. Cell shrinkage due to compaction of organelles in the cytoplasm represents important morphological evidence of apoptosis. (e) At 12 h. hominis.

org 39 .sgmjournals.Metronidazole-induced apoptosis in Blastocystis (a) (b) (c) (d) (e) (f) http://mic.

apoptotic cells exhibit externalization of PS in the early stages of cell death while maintaining membrane integrity (Gatti et al.. Furthermore. Nasirudeen and others In higher eukaryotes. As such. Though agarose gel electrophoresis of B. Falcieri et al. no DNA ladder was noted in apoptotic B. Collins et al. (2003) reported that DNA cleavage in early necrosis is characterized by selective generation of 59 overhangs. 1992. hominis did not result in any apparent ladderlike fragmentation such as that seen in many apoptotic cells. hominis. What accounts for the inconsistency between the TUNEL and agarose gel electrophoresis methods? Apoptosis may have occurred in an asynchronous fashion so that distinct accumulation of DNA fragments was not observed. Mesner et al. 1998). only three showed DNA laddering – despite the immunohistochemical in situ DNA fragmentation detected in all tissue samples. hominis except for two distinct fragments. The role of PS externalization in Blastocystis has yet to be determined. However. Cornillon et al. In our previous studies.. DNA extracts of metronidazoletreated B. hominis grown in normal culture media under normal physiological conditions may contain lipid granules or electron-dense particles. when cells were treated with 10 mg ml21 Rnase A for 3 h at 30 uC. Necrotic B. TUNEL intensity dipped. Didenko et al. The central vacuole of B. hominis cells stained with Annexin V decreased. hominis cells and those exposed to 0?1 % sodium azide (necrosis control) showed only minimal TUNEL fluorescence. (1992) showed that DNA degradation. on the exposure 40 of a cytotoxic antibody to B. Barbieri et al. does not proceed to nucleosomal-sized fragments but rather results in 50–300 kb range DNA fragments which do not readily generate a characteristic ‘ladder’ pattern during agarose gel electrophoresis. In mammalian apoptotic machinery. 1997). Hence. hominis up to 9 h. the two bands from the earlier run correspond to contaminating rRNA fragments.. hominis showed low Annexin V staining. hominis (Nasirudeen et al. Linfert et al.. 2001a). falciparum.. Perhaps. hominis cells showed low TUNEL. Furthermore. This decrease in Annexin V staining could be largely due to the release of apoptotic bodies where part of the cell membrane might be lost in the process (Nasirudeen et al. In apoptotic chloroquine-sensitive P. 1994. hominis cells. 2001a). TUNEL detects 39-OH groups at the end of single-strand and double-strand DNA cuts.. Hirata et al. At 9 h exposure to metronidazole. Membranebound apoptotic bodies containing portions of the fragmented nucleus and an array of intact organelles such as mitochondria were noted in the central vacuole of metronidazole-treated B. 1994. The appearance of the nucleosomal DNA ladder on an agarose gel was regarded as a hallmark of apoptosis (Collins et al. Another possible reason for the lack of DNA laddering in dying B. these two fragments were absent after electrophoresis (data not shown). we postulated that the central vacuole acts as a repository for the storage of apoptotic bodies during apoptosis (Nasirudeen et al. hominis DNA did not show typical ladder-like DNA fragmentation pattern. Darzynkiewicz et al. A. DNA laddering was reported using autoradiography methods when no visible DNA ladder pattern was seen using conventional agarose gel electrophoresis (Picot et al. Hence. In this study. PS residues may have already been degraded by cytosolic lipases. while membrane integrity was largely intact. This could be due to secondary necrosis where much of the nuclear material may have been lost in apoptotic bodies. 1992. which may explain the relatively low Annexin V staining in necrotic cells. the cells or the apoptotic bodies could undergo secondary necrosis. The current research provides more evidence for this role.. Flow cytometry data showed increasing TUNEL staining of metronidazole-treated B. 1987. hominis cells could have a similar function in the clearance of apoptotic bodies. externalization of PS is necessary to signal neighbouring immune cells (Adayev et al. But it has also been shown that internucleosomal DNA fragmentation cannot always be regarded as a hallmark of apoptosis since certain cells display morphological and biochemical features of apoptosis without ladder-like DNA fragmentation (Howell & Martz. Normal B. cells were treated with 0?1 % sodium azide for 24 h to induce necrosis. This can easily be detected by Annexin V staining. hominis cells could be the low sensitivity in the method of detection. Vaux et al. Penfold & Provis (1986) reported that most of the cellular debris resulting from cell death of the retina is taken up by adjacent cells rather than by macrophages.. hominis cells had increased 10-fold from ~5 to 50 %. TUNEL indicated that metronidazole-treated cells do indeed undergo in situ DNA fragmentation. 1998). Without proper disposal of dying cells. the externalization of PS residues during apoptotic death of B... Another reason could be that much of the nuclear material may have degraded during the 24 h necrosis induction and therefore have been lost during the washing of cells. but not 39 overhangs.. (1997) demonstrated that of the 21 DNA samples extracted from ischaemic tissue and subsequently electrophoresed. in many cell types. The preservation of membrane integrity and the simultaneous exposure of PS to the outer leaflet of the plasma membrane (in cells treated with metronidazole) clearly demonstrate the apoptotic effect of the drug on B. 2001a). it could be used for attracting neighbouring phagocytic cells. At 12 h posttreatment.A. Another reason for the inconsistency could be that the fragments could have been of larger sizes and hence not resolved in a 2 % agarose gel. 1993. Annexin V-positive B.. At 12 h of metronidazole treatment. the number of B. in vivo.. This may be the reason why necrotic B. M. 1998). 1992. 1992). The immune cells then phagocytose the apoptotic cells and/or apoptotic bodies to minimize damage to the neighbouring healthy cells. In our earlier study. These results suggest that PS exposure precedes the loss of membrane integrity by several hours. But the central vacuole Microbiology 150 ..

Didenko. 1998. B. 795–808. Gobe. S. Del Bino. Williams.. Lassota. as in the case of T. S. E.... N.. G. Am J Pathol 162. Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell death in the control of cell proliferation. in situ fragmentation of nuclear DNA. S. Loyens.. Cristofalo.org eukaryotes may be conserved at the morphological and biochemical levels. 2001). We believe that B. inflammation) and favour overall parasite survival (Knight. (2003). imidazolidinyl urea and 1. 41 . M.... Cornillon. but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia. the metronidazole concentration was 561027 M. In the case of B. Z. O. 1571–1578. 1256–1261. C. (1992). 2002). Idziorek. D.. P. hominis.. J. Currently.. externalization of PS and formation of apoptotic bodies. A. Hotz. 2001b). 2001a) and metronidazole. For example. hominis infections could be attributed to activation of an apoptotic machinery. Al-Olayan. C. & Traganos. Gross. J Cell Sci 107. D.. C. Ngo. F. Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells. Darzynkiewicz.. Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito.sgmjournals. The results of this research lead one to speculate that cell-death mechanisms in B. Mazza.. L. The effects observed by metronidazole treatment suggest that its efficacy in the treatment of B. V.. (1995).. N. 1854–1864. B. namely preservation of plasma membrane integrity. J. A. M. G.. (1992).. P. Inhibition of apoptosis by zinc: a reappraisal. J. Centini. Biochem Pharmacol 63. Andreassi.g.. it has been shown in other systems that a higher drug concentration could induce necrosis (Anselmi et al. Programmed cell death in Dictyostelium. some cells undergo apoptosis in vivo. A. Further detailed research is required to identify molecules involved in apoptotic mechanisms and regulation of this cell-death pathway. & Baskin. necrosis by widely used preservatives: 2-phenoxyethanol. hominis cells to metronidazole treatment (Haresh et al. H. Billant-Mulot. 451–453. R. J. 2691–2704. D.. E. Further research needs to be done to investigate the effects of higher concentrations of the drug so that dose-dependent effects can be studied. Cell Death Differ 2.. G. and (or) an insufficient dose of. REFERENCES Adayev.. J Neurochem 71. Apoptosis in the malaria protozoan. (2002).. P. Gorczyca. & Franceschi. 39 and 59 overhangs in apoptosis.. but only 59 overhangs in early necrosis. P.. C. 2002). Ameisen. Meserole. 1996). Barbieri. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. H. we described the presence and activity of caspase 3-like proteins in B. D. M... M. J. Capri. Interestingly.2-pentanediol. Davis. 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