The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity
David Lloyd2,
Janine C. Harris2,
Sarah Maroulis1,
Giancarlo A. Biagini1,
Robert B. Wadley1,
Michael P. Turner2 and
Michael R. Edwards1
School of Biochemistry and Molecular Genetics and Cellular Analysis Facility, School of Microbiology and Immunology, University of New South Wales, Kensington, Sydney 2052, Australia1
Microbiology Group, School of Biosciences (BIOSI, Main Building), Cardiff University, Cardiff CF10 3TL, UK2
Author for correspondence: David Lloyd. Tel: +44 29 2087 4772. Fax: +44 29 2087 4305. e-mail: lloydd{at}cf.ac.uk
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ABSTRACT
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Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O2. Thus, when exposed to controlled concentrations of dissolved O2 >8 µM, they gradually lose their ability to scavenge O2. In a washed cell suspension stirred under 10% air in N2 (equivalent to 25 µM O2), inactivation of the O2-consuming system was complete after 3·5 h; during this period accumulation of H2O2 (3 µmol per 106 organisms) and oxidation of cellular thiols to 16% of their initial level occurred. Under 20% air (50 µM O2), respiratory inactivation was complete after 1·5 h, and under air (258 µM O2), after 50 min. Loss of O2-consuming capacity was accompanied by loss of motility. Use of the fluorogen 2,7-dichlorodihydrofluorescein acetate indicated that intracellular H2O2 is produced at extranuclear sites. Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), showed that values declined from -134 mV to -20 mV after 4·5 h aeration. Incubation of organisms with 60 µM H2O2 for 10 min gave partial collapse of plasma membrane potential and complete loss of O2 uptake capacity; motility and viability as assessed by DiBAC4(3) exclusion were completely lost after 1 h. Inactivation of the O2-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.
Keywords: hydrogen peroxide, oxidative stress, reactive oxygen species
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N-dicyclohexylcarbodiimide; DiBAC4(3), bis(1,3-dibutylbarbituric acid) trimethine oxonol
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INTRODUCTION
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The flagellate protozoon Giardia intestinalis is a worldwide cause of intestinal infection and diarrhoea, and especially in infants and the elderly, morbidity (Ortega & Adam, 1997
) and mortality (Shukry et al., 1986
). The trophozoite infects the upper jejunum by attachment to the mucosal surface. Waterborne transmission occurs by ingestion of cysts (Craun, 1990
), which survive for periods of months in freshwater ecosystems at low temperatures. Control of contamination of fresh water and supplies of potable water is achieved only with difficulty, due to resistance of cysts to chemical agents (Winiecka-Krusnell & Linder, 1998
), and the probable existence of animal reservoirs (Smith et al., 1995
).
The upper intestinal lining is well supplied with capillaries, and O2 concentration there has been measured at 60 µM (Atkinson, 1980
). G. intestinalis is usually regarded as an anaerobic protozoon, and grown and studied as such in laboratory culture (Meyer, 1976
). However, several observations indicate that this organism is a microaerophile (Paget et al., 1989
, 1993
). Measurement of O2 consumption as a function of dissolved O2 indicates that at low levels (050 µM), the organism is capable of scavenging O2 (apparent Km for O2 6·4 µM for the trophozoite). Above a threshold of 80 µM O2, O2 inhibits its own consumption. Changes in the balance of major fermentation products occur as O2 concentration is increased. Paget et al. (1990)
found that anaerobically, alanine and ethanol were formed in roughly equimolar amounts, ethanol production occurred maximally at 1·0 µM O2, whereas acetate accumulation continued to increase to at least 46 µM O2. The intracellular redox state, as indicated by in vivo measurement of NADH, and the integrity of an EPR-detectable ironsulphur centre are both highly sensitive to traces (<0·1 µM) of O2 (Paget et al., 1993
; Ellis et al., 1993
). Here we aim to define the limits of O2 tolerance of this microaerophilic organism, and to characterize the nature of the structural and functional consequences of accumulation of reactive O2 species.
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METHODS
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Organisms and cultures.
Giardia intestinalis, Portland 1 strain ATCC 30888, originally described by Meyer (1976)
, was obtained from the Queensland Institute of Medical Research. Trophozoites were grown axenically at 37 °C, using TYI-S-33 medium (Keister, 1983
; Edwards et al., 1989
) but with complement-inactivated new-born calf serum at 10% (v/v) Cultures were initiated by inoculating 15 ml medium in polypropylene centrifuge tubes with 4x104 organisms ml-1. A 0·5 ml air headspace was routinely employed. Growth was for 2 d and final cell numbers reached about 107 per tube.
Harvesting.
Tubes were chilled on ice for 20 min and then shaken gently to dislodge adhered trophozoites. Cell numbers were measured by counting on a haemocytometer slide. Cell suspensions were centrifuged at 1000 g (3000 r.p.m.) for 4 min at room temperature in a bench centrifuge at room temperature. After washing once with phosphate-buffered saline (pH 7·1, PBS) and recentrifugation, organisms were finally resuspended in PBS or in 0·31 M mannitol solution (as indicated) and kept at 4 °C.
Measurements of O2 consumption rates.
Cell suspensions (2·2x10-6 organisms ml-1), 2 ml total volume (1:1) in PBS were incubated at 37 °C in a closed O2 electrode vessel (Rank) with magnetic stirring (200 r.p.m.). Where incubations were performed at low O2 concentrations, a stainless-steel open O2 electrode system fitted with a Teflon-membrane-covered O2 electrode (Radiometer) (Lloyd et al., 1979
) was employed. A digital gas mixer (Lundsgaard & Degn, 1973
) was used to mix 1% or 5% O2 with N2; after humidification by passage over moist filter paper, the gas mixture was passed over the surface of the stirred liquid vortex (stirring at 500 r.p.m.), enabling the O2 tension to be maintained at desired levels. Addition of substrates (e.g. glucose) or of inhibitors were made through a septum. To obtain the transfer constant, k, the value of t1/2 for equilibration of buffer upon switching the gas phase from 1% O2 to N2 was determined, then k was calculated from the relationship k=loge2/t1/2.
Respiration rates (Vr) were calculated from Vr=k(TG-TL), where TG is the O2 concentration in the gas phase, and TL that in the liquid phase.
O2 concentrations in PBS were calculated from the air-saturation value for air at 37 °C (250 µM O2).
Measurement of cellular swelling.
Changes in cell volume were monitored by following the time course of absorbance change at 550 nm.
Confocal laser-scanning microscopy.
A Bio-Rad MRC confocal system attached to a research microscope (1024-Leica DMRB) was used with an argon-krypton air-cooled laser at 448 nm. Images were obtained with ax63 oil-immersion objective (NA 1·38). Section thickness was 5·5 µm. The 0·3 W laser was used at 10% power to minimize photobleaching. Unless otherwise stated, organisms were washed and resuspended in 0·31 M mannitol before observation using FITC filters. Images were acquired on Zip disc and printed using an Epson 750 colour printer.
Flow cytometry.
Forward narrow-angle light scatter, side-scatter and fluorescence were measured using a flow cytometer (Multi-Laser Sorter, MoFlo Cytomation) fitted with a Cicero or a Cyclops Summit version 2 operating software. Fluorescence of bis(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] was measured using a water-cooled 200 mW 488 nm argon-ion laser (Coherent I-90). An interference filter (D530/540) was used for emitted wavelengths (Chrome Technology), together with a Hammamatsu Hybrid Photodetector (no. 957-06). The characteristics of 20000 organisms were accumulated routinely.
Flow cytometric measurement of plasma membrane potential.
Plasma membrane potential was measured by flow cytometry (Krasznai et al., 1995
), using the negatively charged fluorescent membrane potential indicator dye DiBAC4(3). This dye distributes across the cytoplasmic membrane, according to the Nernst equation.
A calibration curve of fluorescence intensity (i.e. channel number) measured from stained cells vs extracellular dye concentration allows evaluation of membrane potential in mV using live cells by comparison with those in depolarized state. Rather than employing fixed organisms (Emri et al., 1998
), heat treatment (>80 °C) for 3 min was employed in order to produce a suspension with zero plasma membrane potential.
Transmission electron microscopy.
Washed G. intestinalis cells were fixed at 4 °C for 1 h in 0·1 M cacodylate buffer (pH 6·9) containing 1% paraformaldehyde and 2% glutaraldehyde. They were post-fixed with phosphate-buffered 1% OsO4 at 4 °C for 1 h, then dehydrated with successive washes of ethanol: 50%, 70%, 90% at 4 °C and two washes of 100% at room temperature. The cells were embedded in Spurr resin. Ultrathin sections were obtained with an LKB Ultratome III and mounted onto 0·5% Pioloform (in chloroform) coated copper grids. The sections were stained with aqueous uranyl acetate and lead citrate. Grids were analysed using a JEOL 1210V transmission electron microscope.
Assay methods.
Thiols were estimated after reaction with Ellmans reagent (5,5'-dithiobis-2-nitrobenzoic acid) at 412 nm (
412=14700 l mol-1 cm-1) (Ellman, 1959
). Hydrogen peroxide was measured in a closed O2 electrode using catalase.
Materials.
Toluidine blue and rose bengal (tetraiodotetrachlorofluorescein) were gifts from Mr Till Böcking and Dr Kevin Barrow.
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RESULTS
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Survival of organisms in the presence of O2
When washed suspensions of G. intestinalis in PBS (pH 7·1) were stirred under a gas phase of air, O2 demand decreased until inactivation of the O2 consumption system was complete after 50 min (Fig. 1a
), when dissolved O2 reached the air-saturated value. Accumulation of H2O2 continued for the duration of the experiment (195 min), to reach 4 µmol per 106 organisms. Under 4% O2 in N2, inactivation took 75 min (Fig. 1b
), whereas under 2% O2 in N2 it took 140 min; under 1% O2 in N2, O2 uptake was still measurable after 4 h. Fig. 1(c)
shows the accumulation of H2O2 and the decrease in total cellular thiols (conversion from thiol to disulphide forms) when the organisms were stirred under 2% O2 in N2. Cessation of O2 consumption under all conditions of exposure to inhibitory O2 concentrations preceded loss of membrane potential (see below), flagellar movement and cell motility. These changes were irreversible; restoration of anaerobiosis did not lead to recovery of cellular function.
Structural changes under O2
Fig. 2(a)
shows a bright-field image of a population of washed G. intestinalis trophozoites in the presence of 1 µM DiBAC4(3). The same field examined for fluorescence (excitation 490 nm, emission 519 nm; Fig. 2b
) indicates that almost all the organisms exclude the fluorophore; those that do not are damaged. After exposure of organisms to O2 almost all the cells still excluded the fluorophore; again those that did not were damaged (not shown). Storage at 4 °C for 18 h (Fig. 2c
) gave a population that was completely DiBAC4(3) permeable. Organisms stirred under 5% air remained impermeable to DiBAC4(3) for more than 6 h, whereas under 10% air after 4·5 h many organisms became fluorescent. Confocal laser-scanning microscopy (Fig. 2d
) indicated that DiBAC4(3) had permeated the plasma membrane and the flagella. Under 100% air, DiBAC4(3) penetration was rapid, occurring in less than 1 h. Heat-killed organisms also became completely DiBAC4(3) permeable (not shown). Exposure to air for 10 min (Fig. 2e
, f
) or incubation with 60 µM H2O2 for 10 min (Fig. 2g
) gave organisms in which 2,7-dichlorodihydrofluorescein became oxidized to give fluorescence in the cytosol. The distribution of the fluorescence suggested that externally applied H2O2 also gave fluorophore oxidation in peripheral vesicles, which was not the case when organisms were oxidatively stressed in air.


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Fig. 2. Micrographs of G. intestinalis incubated with 1 µM DiBAC4(3) (ad) or with 2',7'-dichlorodihydrofluorescein diacetate (eg). (a) Bright-field illlumination. (b) Fluorescence (same field as in a). (c) Fluorescence after storage at 4 °C for 18 h. (d) Confocal laser-scanning fluorescence image of an organism exposed to air for 4·5 h. (eg) Organisms incubated for 10 min in air (e, f; ventral and dorsal aspects respectively), or in 60 µM H2O2 (g).
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Flow cytometry of organisms exposed to O2
Measurement of fluorescence of DiBAC4(3)-treated organisms showed that the population behaved uniformly towards agents that depolarized the plasma membrane potential and that oxidative stress, produced by exposure to air or by externally added H2O2, also had this effect. Fig. 3
shows two typical experiments. Live cells did not take up the anionic fluorophore, whereas heat-killed organisms were highly DiBAC4(3) permeable and hence were counted in channels indicative of high fluorescence intensities. Other treatments known to affect ion transport processes and hence lead to partial collapse of the potential across the plasma membrane resulted in fluorescence emission values intermediate between those of active organisms and the completely depolarized dead cells. Cells aerated for 1 h (Fig. 3a
, panel 5) and those treated with 60 µM H2O2 for 5 min (Fig. 3b
, panel 2) also showed increased permeability to DiBAC4(3). Plots of forward light scatter (a measure of cell size) vs side scatter showed that as cells became damaged by O2, they also progressively increased in volume (data not shown). Heat-killed organisms gave lower forward scatter signals than live ones. Pulse-width measurements showed that cell doublets or aggregates were rare for live, damaged or dead organisms.

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Fig. 3. Flow cytometric analyses of DiBAC4(3)-treated G. intestinalis. (a) 1, Control (live organisms). 26, Treated cells: 2, 10 µM CCCP; 3, 1 µM gramicidin; 4, 100 µM DCCD; 5, aeration for 1 h; 6, heat-killed (>80 °C for 5 min). Incubations were with 0·31 M mannitol/1 µM DiBAC4(3) for 30 min. (b) 1, Control (live cells). 24, Treated cells: 2, 60 µM H2O2 for 5 min; 3, 1 µM gramicidin; 4, heat-killed (>80 °C for 5 min). Organisms were incubated in 0·31 M mannitol/0·5 µM DiBAC4(3) for 30 min. FL1 and FL2 represent the fluorescence emission in channel 2, at 530540 nm.
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Calibration of fluorescence intensity (channel no.) against DiBAC4(3) concentration provides a means of measuring plasma membrane potential (Krasznai et al., 1995
; Emri et al., 1998
). Heat-killed organisms (with zero membrane potential, used as a control) were taken to have an intracellular fluorescence equal to that of dye in the suspending medium. Fig. 4
shows that live organisms when suspended in 0·31 M mannitol had a plasma membrane potential of -134±3 mV (n=6). Exposure to 5% air for 4·5 h decreased this value to -100 mV, whereas under 100% air, G. intestinalis became almost completely depolarized in 4·5 h. Effects of some inhibitors are also shown: the most effective (albeit at high concentration, 100 µM), was the proton-pumping ATPase inhibitor DCCD. Gramicidin (1 µM), an ionphore acting to increase membrane permeability to H+, K+ and Na+, or the protonophore CCCP (10 µM) were both highly efficient at diminishing potentials.

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Fig. 4. Plasma membrane potentials of G. intestinalis as determined by DiBAC4(3) permeability. Determinations were made over a range (0·252·0 µM) of external DiBAC4(3) concentrations.
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Oxygen consumption measurements
The effects of O2 exposure could be mimicked by addition of 60 µM H2O2 to washed cell suspensions in PBS: Fig. 5(a)
shows its immediate inhibitory effect on O2 consumption. That
OH is produced when G. intestinalis is treated with H2O2 is indicated by protective effects of
OH radical quenchers (50 mM sodium benzoate or 50 mM mannitol); ferric nitrilotriacetic acid accentuates H2O2 inhibition. Similar results were obtained with a well-washed non-proliferating suspension of G. intestinalis in a reactor open for gas flow, under an atmosphere of defined low O2 value (Fig. 5b
).

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Fig. 5. O2 consumption by G. intestinalis; inhibition by added H2O2. (a) O2 depletion in a closed vessel fitted with an O2 electrode. Total reaction volume 1·1 ml, stirring at 200 r.p.m., 2·2x106 organisms ml-1. Numbers on the trace are nmol min-1 per 106 organisms. (b) Steady-state dissolved O2 in a vessel open for gas flow. Total reaction volume 6 ml, gas phase, 0·5% O2 in N2 stirring rate 200 r.p.m., 106 organisms ml-1.
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When the organism was illuminated (400 µmol m-2 s-1) during incubation with 2·5 µM toluidine blue or 10 mm rose bengal under 5% air in N2 (=12·5 µM O2), almost complete inactivation of the O2-consuming system was observed within 45 min; after this time organisms were completely immobilized, and for rose bengal, they also became dye-permeable. Similar incubations under anaerobiosis in the light gave no cytotoxic effects.
Electron microscopy
All of the main distinguishing features of G. intestinalis can be seen in the controls shown in Fig. 6(a
, b
), i.e. the ventral disc composed of numerous microtubules, dorsal ribbons and cross-links with its rigid lateral crest, overlying ventrolateral flange and supporting marginal plates or striated bodies. The two equivalent nuclei lie lateral to the kinetosomal complex consisting of eight kinetosomes in four pairs. In the horizontal cross-section (Fig. 6b
) the ventral and posteriolateral axonemes are visible along with the caudal axonemes. The anterior axonemes cannot be seen at this level but the anterior flagella can be seen external to the cell. All axonemes and flagella show the typical 9+2 microtubule arrangement. The vertical section (Fig. 6a
) shows how the anterior axonemes course anteriorly in the cell before crossing and moving posteriorly to emerge at the cells widest point. These axonemes are accompanied by the striated bodies. The microtubules of the median bodies can be seen in their disorganized array. Numerous peripheral vacuoles lie below the plasmalemma. The cytoplasm stains dark owing to the presence of numerous stored granules, e.g. glycogen.


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Fig. 6. Electron micrographs of G. intestinalis Portland-1. (a) Vertical section through the ventral surface of a control trophozoite. (b) Horizontal section at the level of the nuclei through a control trophozoite. (c) Vertical section through the ventral surface of a trophozoite incubated with 60 µm H2O2 for 30 min. (d) Horizontal section through a trophozoite incubated with H2O2 for 30 min. (e, f) Sections through trophozoites that have been aerated for 1 h. (g, h, i) Sections through trophozoites that have been aerated for 2 h. (j) Section through a trophozoite that has been aerated for 3 h. N, nuclei; P, peripheral vesicles; S, striated bodies; D, ventral disc; K, kinetosomal complex; F, funis, M, median body microtubules; A, axonemes; C, lateral crest; V, ventrolateral flange; AF, anterior flagella; MLB, multilamellar body; Cy, cytoplasm.
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Incubation with 60 µM H2O2 for 30 min brought about rapid morphological change and cell breakdown (Fig. 6c
, d
). Most of the cells were ghostlike, with no dark-staining material in the cytoplasm and condensation within the nucleus. Numerous peripheral vacuoles scattered beneath the plasmalemma on the dorsal surface were visible. These appeared to converge and burst the cell. The ventral disc, axonemes and kinetosomes remained intact, indicating that oxidative stress has no evident impact on microtubule structure.
After 1 h aeration, the bulk of the cells maintained the pyriform structure, but some cells became swollen and misshapen (Fig. 6e
). In all cells the ventral disc, its rigid lateral crest and ventrolateral flange were intact; no fragmentation of the disc was visible. The nuclei were no longer spheroid but appeared to have several cytoplasmic projections, and some had associated multilamellar bodies (Fig. 6f
). The axonemes were intact and were still located centrally within the cell. The misshapen swollen cells had large cytoplasmic vacuoles and the dark granule-filled cytoplasm was now very sparse. They appeared ghostlike. In these cells the ventral disc was still intact.
After 2 h aeration the number of swollen and misshapen cells had increased, yet many cells still remained pyriform in structure (Fig. 6g
, h
, i
). Many nuclei had associated multilamellar bodies (Fig. 6h
). The ventral discs, lateral crests and ventrolateral flanges were still intact. The cytoplasm was less dense and did not stain as darkly as previously. The number of peripheral vacuoles had increased.
After 3 h aeration only a few cells remained in the intact pyriform shape. Many cells had completely broken down and cellular debris abounded. The cytoplasm stained only weakly, with very few dark-staining granules remaining. Most cells were not recognizable as Giardia (Fig. 6j
). The nuclei had broken down and their chromatin had condensed. The ventral disc and axonemes were still intact, suggesting again that oxidative stress does not affect the microtubule structure within the cell.
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DISCUSSION
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G. intestinalis has previously been shown to have a limited capability for withstanding exposure to dissolved O2 (Paget et al., 1989
). In those experiments where the O2 dependence of O2 uptake were investigated, the concentration of dissolved O2 was increased from an undetectable level to 5% air saturation in a series of steps of 20 min each. Those data provide indications on apparent Km values and of a threshold above which O2 became inhibitory. In the present investigation, we have systematically investigated the time- and concentration-dependence of the O2 effects during long-term maintenance of steady-state levels. Oxidative damage was shown, not only to the electron-transport systems responsible for O2 reduction, but to cell functions across a wide range. Thus damage to the plasma membrane, as indicated by the progressive loss of ability to exclude the anionic voltage-sensitive dye DiBAC4(3) (Lloyd & Hayes, 1995
), led to swelling of organisms and loss of the regulatory volume control process, described in fully functional organisms (Park et al., 1995
). Sites known to be O2-inhibited in G. intestinalis include a (4Fe4S) ironsulphur cluster (Ellis et al., 1993
) and redox-active thiols involved in volume regulation (Park et al., 1995
). The fact that these effects could also be produced almost immediately by 60 µM H2O2 suggests that exposure to inhibitory levels of O2 results in accumulation of this partial reduction product. The Fenton HaberWeiss reaction is then free to produce
OH from H2O2 and 
, especially as this organism contains no detectable superoxide dismutase (Smith et al., 1988
), peroxidase or catalase (Brown et al., 1995
). Enhanced loss of functional activity in the presence of ferric nitrotriacetic acid (Aruoma et al., 1989
) and diminished inactivation in the pressure of the
OH radical scavengers, sodium benzoate or mannitol (Halliwell, 1978
) suggests that
OH, a radical known for its extremely damaging reactivity (Halliwell & Gutteridge, 1989
) may be an important intracellular oxidant in these experiments. The O2-dependent production of photo-emissive species in Giardia lamblia has been demonstrated (T. Paget & D. Lloyd, unpublished data). These species, observed as low-level chemiluminescence in organisms metabolizing glucose, could be greatly enhanced in the presence of the redox-cycling naphthoquinone menadione. The fact that the oxygen-consuming redox chains of organisms can be damaged by 1O2-generating systems, the photodynamically activated dyes toluidine blue and rose bengal (Krinsky, 1979
), supports the suggestion (Lamberts & Neckers, 1985
) that the menadione sensitivity of G. lamblia can also be accounted for by free-radical damage by reactive oxygen species. Thus the anti-giardial effects of a redox-cycling naphothquinone are produced by an amplification of the effects of O2 damage. The anti-giardial effects both of menadione, and of H2O2 reported here, warrant further investigation as a means of control of this important human pathogen.
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ACKNOWLEDGEMENTS
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We are grateful to Dr Kevin Barrow and Mr Till Bocking for advice on singlet oxygen generation. The work was in part supported by a travel grant from the Royal Society to D.L.
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Received 7 June 2000;
revised 7 August 2000;
accepted 21 August 2000.