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Oxidative stress-induced cell cycle blockage and a protease-independent programmed cell death in microaerophilic Giardia lamblia.
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PMID:  19920926     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Giardia lamblia is a microaerophilic human gastrointestinal parasite and considered as an early-diverged eukaryote. In vitro oxidative stress generation plays a significant role in cell cycle progression and cell death of this parasite. In the present study hydrogen peroxide, metronidazole, and a modified growth medium without cysteine and ascorbic acid have been chosen as oxidative stress-inducing agents. Cell cycle progression has been found to be regulated by different types of oxidative stresses. Apoptosis is not an established pathway in Giardia, which is devoid of ideal mitochondria, but in the present investigation, apoptosis-like programmed cell death has been found by the experiments like AnnexinV-FITC assay, DNA fragmentation pattern, etc. On the contrary, Caspase-9 assay, which confirms the caspase-mediated apoptotic pathway, has been found to be negative in all the stress conditions. Protease inhibitor assay confirmed that, even in absence of any proteases, programmed cell death does occur in this primitive eukaryote. All these results signify a novel pathway of programmed suicidal death in Giardia lamblia under oxidative stress. This is the first demonstration of protease-independent programmed cell death regulation in Giardia exclusive for its own specialties.
Authors:
Esha Ghosh; Arjun Ghosh; Amar Nath Ghosh; Tomoyoshi Nozaki; Sandipan Ganguly
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-09-21
Journal Detail:
Title:  Drug design, development and therapy     Volume:  3     ISSN:  1177-8881     ISO Abbreviation:  Drug Des Devel Ther     Publication Date:  2009  
Date Detail:
Created Date:  2009-11-20     Completed Date:  2012-09-10     Revised Date:  2013-05-23    
Medline Journal Info:
Nlm Unique ID:  101475745     Medline TA:  Drug Des Devel Ther     Country:  New Zealand    
Other Details:
Languages:  eng     Pagination:  103-10     Citation Subset:  -    
Affiliation:
Division of Parasitology, National Institute of Cholera and Enteric Diseases, P 33 Cit Road, Beliaghata, Kolkata,West Bengal, India.
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Journal Information
Journal ID (nlm-ta): Drug Des Devel Ther
Journal ID (publisher-id): Drug Design, Development and Therapy
ISSN: 1177-8881
Publisher: Dove Medical Press
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? 2009 Ghosh et al, publisher and licensee Dove Medical Press Ltd.
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collection publication date: Year: 2009
Electronic publication date: Day: 21 Month: 9 Year: 2009
Volume: 3First Page: 103 Last Page: 110
ID: 2769235
PubMed Id: 19920926
Publisher Id: dddt-3-103

Oxidative stress-induced cell cycle blockage and a protease-independent programmed cell death in microaerophilic Giardia lamblia
Esha Ghosh1
Arjun Ghosh1
Amar Nath Ghosh2
Tomoyoshi Nozaki3
Sandipan Ganguly1
1 Division of Parasitology
2 Division of Electron Microscopy, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, West Bengal, India
3 Division of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan
Correspondence: Correspondence: Sandipan Ganguly, Division of Parasitology, National Institute of Cholera and Enteric Diseases, P 33 CIT Road, Scheme XM, Beliaghata, Kolkata ? 700010, West Bengal, India, Tel +91 33 2363 3855, Fax +91 33 2370 5066, Email sandipanganguly@gmail.com

Introduction

Living aerobic organisms, from prokaryotes to complex eukaryotes, have developed elaborate sequences of adaptive mechanisms to maintain oxygen homeostasis and equilibrium.1 Any deviation from homeostasis, or physiological change in oxygen pressure, is recognized as an exposure to oxidative stress.2Giardia lamblia is an intestinal protozoan parasite causing malabsorption and diarrhea (giardiasis) in human. It is an early diverging eukaryote with unicellular, binucleated, flagellated structure, without ideal mitochondria, golgi complex or peroxisomes.3,4 Measurement of O2 consumption as a function of dissolved O2 indicates that at low levels (0?50 ?M), the organism is capable of scavenging O2 (apparent Km for O2 6.4 ?M for the trophozoite). On the contrary, in human gut cell lining, Giardia resides in a higher oxygen tension where the O2 concentration has been measured at 60 ?M.5 This fact has become more interesting as the conventional enzymes for detoxifying reactive oxygen species (ROS) (viz. superoxide dismutase [SOD], catalase, peroxidase, glutathione reductase) are not found in Giardia.6 Change in the oxygen environment has played the key role in the evolution of aerobic organisms from anaerobes. In case of Giardia, it is also evident that change in oxygen pressure has achieved an important role in its survival strategy as well as the evolution and adaptation of this parasite.

In the present study, three conditions have been chosen to generate oxidative stress in the trophozoites in vitro. First, hydrogen peroxide (H2O2) is a very well known chemical reagent that can generate free oxygen radicals very easily.7 Secondly, metronidazole is a commonly used drug against giardiasis in the most developing countries. Its mode of action involves free nitro radical generation within the organism.8 Third one is a modified medium that is devoid of cysteine and ascorbic acid. Cysteine has been found to protect G. lamblia trophozoites from thiol-blocking reactants, indicating a role of reducing agent for protection of crucial thiol groups. Ascorbic acid also protects the trophozoites under high pO2.9 These three conditions would give us an idea about the effects of different modes of oxidative stresses on Giardia cells. Cell cycle analysis using flow cytometer has been carried out to check the effects of oxidative stress on survival of parasitic cells. The changes in whole cell morphology have been studied in each case using transmission electron microscopy (TEM). As mitochondria regulates the caspase-dependent apoptotic cell death pathway in higher eukaryotes and no ideal mitochondria has been reported in Giardia, it was our endeavor to identify whether a programmed cell death (PCD) is possible in this early divergent eukaryote. Several cell death assays have been performed to verify whether the mode of death is apoptotic or necrotic. Protease inhibitor assay has also been performed to check if other proteases like serine/cysteine proteases, calpains, fragmentin/granenzymes, etc are playing any role in parasitic death as they are reported in higher eukaryotes.10

Oxygen concentration always plays a vital role in the survival strategy and commitment of death from lower groups of organisms to higher eukaryotes. With the changes in global oxygen concentration from one era to another along thousands of years, lives on earth have evolved themselves from obligatory anaerobes to obligatory aerobic system via several facultative and mutual adaptations. Currently, the detail mechanism is not clear on how these severe changes occurred gradually with time. Studies on oxidative stress regulation in Giardia and similar early branching eukaryotes (EBE) may shed some new ideas about these changes.


Materials and methods
Giardia culture maintenance

Giardia lamblia Portland I was maintained axenically in TYIS-33 medium at 35.5 ?C and sub-cultured at each 48 h.

Standardization of the conditions generating oxidative stress

Dose and time kinetics of the three different oxidative stress-generating conditions (as described above) have been standardized following the IC50 values as reported previously.7,11 Finally from the standardized data, 0.1 ?M H2O2 and 1 ?g/ml metronidazole have been administered in all the experiments mentioned below.

Cell fixation and staining for cell cycle analysis in flow cytometry

Two ? 106 cells were harvested from each culture sets (control and three experimental sets) and the cell pellet were taken by centrifugation at 2000 rpm for 10 min. Cells were washed with phosphate buffered saline (PBS) and treated with methanol for fixation and then in 1% Triton X for membrane permeablization. RNase treatment was carried out prior to propidium iodide (PI) staining of the cells. It was incubated on ice and then used for cell cycle analysis using flow cytometer (FC 500; Beckman Coulter, Miami, FL, USA).

Cell death assays
Cell morphology study by electron microscopy

Control and all sets of experimental samples were harvested (~106 trophozoites in each case), prefixed with 3% gluteraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and post fixed in 1% osmium tetraoxide in 0.1 M sodium cacodylate buffer (pH 7.4). The post-fixed cells were block stained in 1% uranyl acetate and then treated with graded series of ethanol and subsequently embedded in agar 100 resin (Agar Aids, Stansted, UK). Ultra thin sections were cut in a Leica Ultracut UCT ultramicrotome and stained with uranyl acetate and lead citrate. Sections were examined in a Tecnai 12 Bio-twin transmission electron microscope (FEI, Hillsboro, OR, USA).

Annexin V-FITC assay

This experiment was performed using Annexin V-FITC Kit (Beckman Coulter), following the manufacturer?s protocol. Briefly, cell samples (Control cell as well as oxidative stressed cells) were washed with chilled PBS and cell pellet was resuspended in 1 ? binding buffer and kept on ice. Annexin V-FITC solution was added and incubated on ice in dark. Cell preparation was analyzed using confocal microscope (LSM 510, Meta; Carl Zeiss, Thornwood, NY, USA).

FITC-PI staining

For this experiment the cells were similarly treated with Annexin V-FITC solution (Beckman Coulter) as per manufacturer?s protocol as mentioned above. Along with that, PI solution was also added and incubated on ice in the dark. This cell preparation was analyzed for its immunofluorescence using confocal microscope and flow cytometer.

DNA fragmentation

Permeabilized cells (as prepared above) were treated with PI to observe the DNA damage in nucleus under confocal microscope.

DNA laddering (degradation)

Control cells and stressed cells were harvested and resuspended in digestion buffer (10 mM EDTA, 50 mM Tris, 0.5% SDS Sarcosine, pH = 8.0) containing 0.5 mg/ml proteinase K. Samples were incubated at 37 ?C for 1 h after adding DNase-free RNase (0.1 mg/ml). After phenol?chloroform treatment, salt preciptation and 70% ethanol wash, pellet was air dried and resuspended in autoclaved triple distilled water and checked in a 1% agarose gel stained with ethidium bromide.

Caspase-9 assay (Colorimetric)

This assay was performed using Caspase-9 Assay Kit (Calbiochem?, San Diego, CA, USA) following manufacturer?s protocol. Briefly, all the control and stressed cell pellets were resuspended in cell lysis buffer provided with the kit and incubated on ice for 10 mins. It was then centrifuged and the supernatant (cytosolic extract) was collected in a tube, kept on ice. After measuring protein concentration it was dissolved in 50 ?l cell lysis buffer. Then reaction buffer with 10 mM DTT was added to each sample. Finally LEHD-pNA substrate (200 ?M final concentration) was added, incubated at 37 ?C and samples were read at 400?405 nm.

Protease inhibitor assay

Mixture of several protease inhibitors (a broad range protease inhibitor; Roche, Nutley, NJ, USA) were added to the culture and incubated for 1 h. Small water soluble molecules of protease inhibitors were absorbed by the cells along with medium. Then the cells were treated with oxidative stress producing agents. Annexin V-FITC assay and DNA-laddering assay were repeated to check if programmed cell death occurred in absence of proteases.


Results and discussion
Cell cycle analysis in flow cytometry

In case of Giardia, normal trophozoites of G1 phase contain 4 N chromosome.12 Hence, in control set, maximum numbers of cells lie in G0/G1 phase (Figure 1i). In case of metronidazole, as observed in flow cytometer, the cell cycle was arrested near S-phase (Figure 1ii). Metronidazole is known to exert its toxic effect through unstable intermediate products (nitro and nitroso-free radicals).14 The reactive products also interact with DNA, causing DNA damage, strand breakages, and cross-linking in the nucleic acid and the release of nucleotide phosphates. Hence in our study, the growth arrest of trophozoites near S-phase of cell cycle is due to a similar action of metronidazole. It probably inhibits the DNA segregation and hence prevents the completion of cell division. Peroxides have been found to induce a G1 checkpoint response (Figure 1iii). It suppresses S phase entry by inhibiting cyclin E/Cdk2 activity in a dose-dependent manner.13 In this experiment, we have also found that the maximum number of cells lie in sub-G1 stage. It might also be an indication of apoptotic cell death due to peroxide application. For cystein-ascorbate deprived modified medium, most of the cells move to apoptotic death, but a significant amount has a tendency to increase their DNA content (Figure 1iv).

Cell death assays
Cell morphology study by electron microscopy

Oxidative stress induced cells have shown apoptotic features like membrane blebbing and increased vacuolation in comparison to control cells under transmission electron microscopy (Figure 2).

Annexin V-FITC assay

Externalization of phosphatidyl serine (PS) is one of the indicators of apoptosis in the eukaryotic cells. Fluorescein isothiocyanate (FITC)-tagged annexin can bind with the PS when it comes outside the membrane and the fluorescence signal can be measured in a fluorescent microscope. A characteristic green ring formation can be observed in the membrane of an ideal apoptotic cell. In our study we have used this assay to check the mode of death in stress-induced cells. Normal live cells do not show any ring formation where as in case of all the experimental sets treated with different oxidative stress producing agents revealed a clear ring formation in their outer membrane part (Figures 3A, B, D, and F) confirming the mode of cell death to be apoptotic.

FITC-PI staining

Cells stained with FITC and PI can be targeted for observing their viability and mode of death in a flow cytometer. The gated region in the flow cytometer denotes the type of cells according to their location in the four coordinates. Live cells do not take the stains and lie in the left hand bottom panel where as apoptotic cell population taking FITC stain lies in right hand bottom panel. All the secondary necrotic cells taking the PI stain lie in the right hand upper panel. In our experiment, control live cells did not take any type of stains and consequently lied in the left hand bottom panel (Figure 4A). All the experimental sets showing cell populations in the right hand bottom panel indicate the intake of FITC stain specific for apoptotic death (Figures 4B, C, and D).

DNA degradation

Cell nucleus takes the PI stain as this dye intercalates in the DNA. In normal live cells, the two nucleus of Giardia can be easily identified after PI staining (Figure 5A). DNA degradation is another hallmark for apoptotic type of cell death. Hence, after the onset of apoptosis the genetic material gets dispersed throughout the cell and dispersed red patches of the dye can be visualized under fluorescent microscope. In all the experimental sets treated with different stress-producing agents, DNA degradation has been observed (Figures 5B, D, and F).

DNA laddering (fragmentation)

Cells undergoing apoptosis show characteristic morphological and biochemical features. The biochemical marker for apoptosis is the degradation of the genomic DNA, an irreversible event for the programmed cell death and occurs before changes in plasma membrane permeability. DNA fragmentation (mainly in lower molecular weight region) is the characteristic for apoptosis in some lower eukaryotes. Distinct DNA laddering is absent in these organisms. Compared with the control one, all the test samples are showing sheared fragmented DNA in low molecular weight region (Figure 6).

Colorimetric caspase-9 assay

Higher eukaryotes having developed apoptotic machinery undergo caspase dependent pathway to carry out apoptosis. Caspase 9 initiates the signaling pathway. It is expected that in a programmed cell death, colorimetric caspase-9 assay will produce a positive result confirming caspase-dependent apoptotic pathway. In our study, a negative result was obtained (Table 1) indicating that the programmed cell death in this parasite is devoid of any caspase-signaling pathway.

Protease inhibitor assay

In some cases where caspase is not activated in programmed cell death, other proteases such as serine/cysteine proteases, calpeins, or granenzymes, etc get involved in the pathway. So, protease inhibitor assay was performed to check whether other proteases except caspase control the programmed cell death in Giardia. The cells were treated with a large-scale protease inhibitor cocktail before applying the stress-producing agents. Surprisingly, it was found that in absence of proteases, the trophozoites still could undergo PCD. The result has been verified by Annexin V assay (Figures 3C, E, and G) and DNA degradation assay (Figures 5C, E, and G). Characteristic green fluorescent ring formation and scattered PI staining were obtained in each set, respectively.

A similar type of report has been obtained in the case of Trichomonas vaginalis.15 PI has shown typical DNA degradation in the dead cells. But it has been found that caspase or other proteases are not responsible for this type of death. Cells, after incubating with protease inhibitors when undergo stress, still committed death in programmed pathway. This was confirmed by Annexin V test and DNA fragmentation pattern, as before. Two cases in Leishmania and Blastocystis have been reported where also the PCD is independent of caspase pathway or other proteases.16,17 It is possible that these EBE including our model organism G. lamblia have undergone PCD using a novel pathway.


Acknowledgments

This study was supported by a grant from Okayama University Program of Founding Research Centre for Emerging and Reemerging Infectious Disease, Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors would like to thank Prof Y Takeda and Dr GB Nair, NICED, Japan for their constructive comments and suggestions during the study. Authors are also thankful to Debarati Ganguly of Calcutta University for her critical reading and English correction.


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Article Categories:
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Keywords: Giardia lamblia, oxidative stress, reactive oxygen species, programmed cell death, apoptosis, early branching eukaryotes.

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