1 Department of Applied Biology, University of Hull, UK
2 School of Biotechnology and Bio-molecular Sciences, University of New South Wales, Sydney, Australia
3 Department of Biology, Northeastern University, Boston, MA 02115, USA
4 Microbiology Group (BIOSI 1, Main Building), Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
Correspondence
David Lloyd
Lloydd{at}cf.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The mechanism by which metronidazole, one of the most effective of the nitroimidazoles, kills G. intestinalis is not completely understood. Free radical production from nitroimidazoles was demonstrated firstly using liver microsomes (Perez-Reyes et al., 1980) and then in the cattle parasite Tritrichomonas foetus (Moreno et al., 1983
, 1984
). In a variety of microaerophilic and anaerobic protists and bacteria, it has been suggested that drug reduction products are responsible for organism killing; these products include the nitro radical anion generated by electron donation from pyruvate : ferredoxin oxidoreductase (PFOR) via ferredoxin species [e.g. in the hydrogenosomes of trichomonads (Moreno & Docampo, 1985
; Yarlett et al., 1986b
, 1987
)]. Drug free radical generation also leads to the production of reactive oxygen species (ROS) by a redox cycling mechanism. Resistance to metronidazole is known, in some strains of Trichomonas vaginalis, to be due to altered levels of PFOR (Kulda et al., 1993
; Cammack et al., 2003
), altered redox properties of ferredoxin (Yarlett et al., 1986b
) or diminished pools of drug radical anions due to elevated intracellular O2 levels (Lloyd & Pedersen, 1985
; Yarlett et al., 1986a
).
Resistant strains of G. intestinalis are sometimes also encountered as clinical isolates; one characteristic of certain resistant strains is the presence of elevated NAD(P)H oxidase activity (Ellis et al., 1993b). An explanation for this observation (Fig. 1
) involved the diversion of reducing power away from the site at which drug reduction to the toxic free radical species can occur.
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METHODS |
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Harvesting.
After chilling for 20 min in an ice bucket, tubes were inverted several times to dislodge adherent organisms. After counting, cells were harvested at 1000 g (3000 r.p.m.) for 4 min at room temperature in a bench centrifuge (MSE Minor). After washing once with PBS (pH 7·4) and recentrifugation, organisms were finally resuspended in PBS and kept at 4 °C.
Analytical methods.
Absorbance and light scattering measurements (Park et al., 1997) were done in a Beckmann DV 7500 UV/visible spectrophotometer. Oxygen consumption at controlled low O2 concentrations was in a stirred reaction vessel fitted with a Radiometer (Copenhagen) O2 electrode in a Hitachi 557 double-beam spectrophotometer (Degn et al., 1980
) or used in an EMI single photon counting luminometer fitted with a cooled (20 °C) low-background photomultiplier tube (Lloyd et al., 1979
; Biagini et al., 2001
). Gas mixtures were made using pre-purified N2; O2 and solubility tables were used to calculate dissolved O2 (Wilhelm et al., 1977
). H2O2 was measured using microperoxidase (Paget et al., 1987
).
Electron spin resonance (ESR) spectrometry.
Incubation of trophozoites under microaerobic conditions in the cavity of the Varian E109 ESR spectrometer was as described in detail previously (Lloyd & Pedersen, 1985). Organisms, suspended in PBS, were placed in an O2-permeable silicone rubber catheter tube in the presence of 20 mM glucose and 50 mM metronidazole. The O2 partial pressure of the mobile gas phase was fixed at a series of values from 0 to 101 kPa, using a digital gas mixing device (Degn et al., 1980
).
Materials.
Gases were from Air Products. Tryptone was purchased from Becton-Dickinson, and trypicase from BioMérieux. Fetal calf serum was supplied by Gibco-BRL, and Nunclon screw-capped culture tubes were from Life Technologies. All other chemicals were from Sigma-Aldrich-Fluka.
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RESULTS |
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Although metronidazole is one of the most potent anti-giardial agents currently in use (Breccia et al., 1982; Ellis et al., 1993b
) its toxicity towards G. intestinalis cysts is somewhat limited (Paget et al., 1993
, 1998
). Therefore, a comparison between the anti-giardial efficacies of the nitroimidazole (metronidazole) and menadione against both developmental stages of the organism was conducted. Against trophozoites, metronidazole and menadione had LC50 values (µg ml1) of 1·2±0·2 and 0·7±0·2, respectively. Against cysts, the anti-giardial agents had LC50 values of >50 and 1·3±0·3, respectively. Therefore, menadione was much more effective at killing trophozoites and cysts of G. intestinalis than metronidazole.
Incubation of 100 µM menadione with G. intestinalis trophozoites gave changes in UV absorbance (Fig. 2). Thus, under anaerobic conditions (sparging with pre-purified N2), progressive reduction occurred over 25 min (
max shifted from 239 to 226 nm, A274 decreased and A334 increased, spectra 2 and 3). Exposure to O2 gave partial reversal of these changes (spectrum 4). Organisms initially showed accelerated O2 consumption when incubated with menadione under microaerobic conditions (Fig. 3
a), but prolonged exposure to reaction products eventually gave complete inhibition of O2 demand and loss of motility of the trophozoites. Menadione concentration giving 50 % inhibition of O2 uptake was 3 µM. These changes were paralelled by accumulation of H2O2, which could be trapped in the presence of microperoxidase (Fig. 3b
) (Paget et al., 1987
). Organisms exposed to 10 µM O2 produced H2O2 at a basal rate, and the addition of 10 µM menadione gave a sixfold increase during the phase of accelerated O2 consumption. The O2-dependence of H2O2 generation was also demonstrated in this system. In a similar experiment, we monitored low-level chemiluminescence (Lloyd et al., 1979
), characteristic of singlet O2 (Fig. 3c
). In the absence of menadione, photon counts from a concentrated suspension of G. intestinalis trophozoites (4x108 ml1) were less than 50 s1 at 15 µM O2 and were decreased to <10 s1 under an atmosphere of pre-purified N2. Menadione (10 µM) stimulated both O2 consumption and photon emission at 15 µM O2, and switching the mobile gas phase from 0·8 kPa O2 to N2 led to rapid exhaustion of dissolved O2 and decreased chemiluminescence. Re-oxygenation gave an overshoot in light emission before relaxation to the microaerobic steady state.
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DISCUSSION |
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This in turn provides inter-radical reactions (Halliwell & Gutteridge, 1989; Guillen et al., 1997
).
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We demonstrate here that both life cycle stages are killed by menadione (LC50 values of 0·7 µM and 1·3 µM for trophozoites and cysts, respectively). The menadione free radical generates H2O2, as measured by the formation of Complex I of microperoxidase, and probably also singlet oxygen 1O2, as monitored by single photon counting of chemiluminescence. Trophozoites swell and become incapable of regulatory volume control. Menadione is therefore a potent anti-giardial agent active against cysts as well as trophozoites, and merits further studies as a potentially useful compound for chemotherapeutic treatment of infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 October 2003;
revised 24 January 2004;
accepted 5 February 2004.
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