The antioxidant potential of pyruvate in the amitochondriate diplomonads Giardia intestinalis and Hexamita inflata

Giancarlo A. Biaginia,1, Jeong H. Park1, David Lloyd2 and Michael R. Edwards1

School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney 2052, Australia1
School of Biosciences, University of Wales Cardiff, Cardiff CF1 3TL, UK2

Author for correspondence: Giancarlo A. Biagini. Tel: +44 151 7053151. Fax: +44 151 7089007. e-mail: Giancarlobiagini{at}hotmail.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Giardia intestinalis and Hexamita inflata are microaerophilic protozoa which rely on fermentative metabolism for energy generation. These organisms have developed a number of antioxidant defence strategies to cope with elevated O2 tensions which are inimical to survival. In this study, the ability of pyruvate, a central component of their energy metabolism, to act as a physiological antioxidant was investigated. The intracellular pools of 2-oxo acids in G. intestinalis were determined by HPLC. With the aid of a dichlorodihydrofluorescein diacetate-based assay, intracellular reactive oxygen species generation by G. intestinalis and H. inflata suspensions was monitored on-line. Addition of physiologically relevant concentrations of pyruvate to G. intestinalis and H. inflata cell suspensions was shown to attenuate the rate of H2O2- and menadione-induced generation of reactive oxygen species. In addition, pyruvate was also shown to decrease the generation of low-level chemiluminescence arising from the oxygenation of anaerobic suspensions of H. inflata. In contrast, addition of pyruvate to suspensions of respiring Saccharomyces cerevisiae was shown to increase the generation of reactive oxygen species. These data suggest that (i) in G. intestinalis and H. inflata, pyruvate exerts antioxidant activity at physiological levels, and (ii) it is the absence of a respiratory chain in the diplomonads which facilitates the observed antioxidant activity.

Keywords: parasite, oxidative stress, reactive oxygen species, yeast, dichlorodihydrofluorescein diacetate

Abbreviations: SOD, superoxide dismutase

a Present address: Liverpool School of Tropical Medicine, Penbroke Place, Liverpool L3 5QA, UK.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The diplomonads Giardia intestinalis and Hexamita inflata are flagellated protozoa which inhabit O2-limited environments. G. intestinalis is a parasite which colonizes the mucosa of the gastrointestinal tract, causing one of the most common water-borne diseases in humans – giardiasis (Adam, 1991 ). H. inflata is free-living and can be found in the anoxic regions of marine and freshwater environments (Dando et al., 1993 ; Fenchel et al., 1995 ). In addition, parasitic species of Hexamita (which may have free-living stages) have been observed in a variety of vertebrate and invertebrate animals (e.g. Kulda & Nohynková, 1978 ; Buchmann et al., 1995 ). The energy demand in these organisms is met by carbohydrate and amino acid fermentation (Brown et al., 1998 ; Biagini et al., 1998 ). The principal products of glucose metabolism include ethanol, alanine, acetate (also lactate for H. inflata) and CO2 (Brown et al., 1998 ; Biagini et al., 1998 ). Pyruvate is central to this metabolism, and the relative rates of generation of the end-products are influenced by the ambient values for O2 tension (Biagini et al., 1998 ; Paget et al., 1993a ). Both of these organisms lack mitochondria or detectable cytochromes (Brugerolle, 1974 ; Paget et al., 1993b ; Biagini et al., 1997 ) (and hence oxidative phosphorylation), but do, however, have high affinities for O2, comparable to those of aerobic protozoa (Paget et al., 1993b ; Biagini et al., 1997 ). The consumption of O2 has been shown to rise linearly with the surrounding O2 tension, up to a threshold level (30–100 µM O2, depending on the species), above which consumption is arrested due to the formation of reactive oxygen species (Paget et al., 1993b ; Biagini et al., 1997 ; Lloyd et al., 2000 ). The oxygen consumption has been attributed to the activity of NAD(P)H oxidase, and it has been postulated that the subsequent change in the redox state of the NAD(P)H pools affects the relative rates of production of end-products (Brown et al., 1998 ; Paget et al., 1993a ; Biagini et al., 1997 ).

Much of the energy metabolism in diplomonads more closely resembles that of bacteria than that of eukaryotic cells. Examples include the presence of pyrophosphate-dependent glycolytic enzymes (Mertens, 1990 ; Phillips et al., 1997 ), the eubacterial-like pyruvate:ferredoxin oxidoreductase (Townson et al., 1996 ), and the presence of the arginine dihydrolase pathway (Schofield et al., 1990 ; Biagini et al., 1998 ; Dimopoulos et al., 2000 ). The antioxidant defence system in diplomonads is also unlike that found in most eukaryotes. Cysteine replaces glutathione as the major intracellular thiol (Brown et al., 1993 ; G. A. Biagini, D. M. Brown & M. R. Edwards, unpublished observation in H. inflata), and catalase and non-specific peroxidase activities are undetectable (Biagini et al., 1997 ; Brown et al., 1995 ). An Fe-type superoxide dismutase (SOD) has been described for H. inflata (Biagini et al., 1997 ) and, although SOD activity was initially reported in Giardia (Thompson et al., 1993 ), in a more recent study SOD activity was undetectable even after induction with 1,10-phenanthroline (Brown et al., 1995 ). In addition, G. intestinalis contains a thioredoxin-reductase-like disulphide reductase which has the ability to reduce cystine (Brown et al., 1996 ), whereas H. inflata contains an as yet uncharacterized thiol reductase which may be involved in cystine reduction (Biagini et al., 1997 ).

The ability of pyruvate to react with H2O2 (and ), forming acetate and CO2 non-enzymically, has long been known (Holleman, 1904 ). However, whether pyruvate plays a physiological role as an H2O2 scavenger in cells is not clear. In this study, we report on the ability of pyruvate, a central component of diplomonad metabolism, to act as an intracellular scavenger of reactive oxygen species in both G. intestinalis and H. inflata. The contrasting physiological roles of pyruvate in oxidative and fermentative metabolism are discussed.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Organisms and solutions.
The G. intestinalis Portland 1 strain and H. inflata were grown as described previously (Knodler et al., 1994 ; Biagini et al., 1997 ). For experimentation, cells were grown to late-exponential phase, harvested by centrifugation at 650 g for 5 min and resuspended in PBS buffer (pH 7·2) containing 150 mM NaCl, 5 mM K2HPO4 and 1·8 mM KH2PO4. Saccharomyces cerevisiae (a laboratory wild-type strain) was grown in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone and 2% (w/v) D-glucose, in a shaking incubator at 30 °C. For experimentation, cells were grown to mid-exponential phase, harvested by centrifugation at 1000 g for 5 min and resuspended in PBS. Pyruvate (the Na salt), menadione and H2O2 solutions were prepared freshly on the day of experimentation.

Determination of intracellular 2-oxo acid pools in G. intestinalis.
Pre-purification, derivatization and subsequent separation of 2-oxo acids in giardial extracts were based on the methods described by Hayashi et al. (1982) and Liao et al. (1977) . Dry polyacrylamide beads (15 g Bio-Gel P-60) were allowed to swell overnight in distilled water (200 ml). The gel suspension was then added to a 98% hydrazine hydrate solution (120 ml) and mixed for 6 h at 50 °C. At the end of the reaction period, the gel was washed free of hydrazine with 0·1 M NaCl and suspended in a storage solution containing 0·2 M NaCl, 0·02 M Na2EDTA, 0·1 M H3BO3, 5 mM NaOH and 5 µM pentachlorophenol at 4 °C. Cell suspensions in PBS were centrifuged at 10000 g through oil (a mixture of dibutyl phthalate and diiso-octyl phthalate, 4:1, v/v; 1·03 g ml-1) into 0·1 ml 1 M perchloric acid. The perchloric acid extract was placed on ice for 1 h and then centrifuged at 10000 g for 1 min. To 0·1 ml of the supernatant from the perchloric acid extract, 0·2 ml of an internal standard solution of 2-oxo-octanoate (80 µM) together with 1 ml 0·1 M acetic acid and 3 ml 0·1 M NaCl were added. The mixture was then loaded onto a glass column containing the 0·3 ml of hydrazide gel. After elution was complete, the gel was washed five times with 0·1 M NaCl and then transferred to a test tube; o-phenylenediamine solution (2 ml) was added to the gel and this was then incubated at 80 °C for 2 h, after which 0·5 g Na2SO4 was added. The derivatives of the 2-oxo acids were extracted with ethyl acetate, evaporated to dryness under a stream of N2, and the residue was dissolved in methanol. HPLC was carried out with a 250x4 mm Li-Chrosorb RP-8 (Capital HPLC) (5 µm particle size) column. The mobile phase consisted of a 6:4 methanol:water mix operating at a flow rate of 1 ml min–1 at 35 °C. The overall recovery of 2-oxo acids measured using radiolabelled pyruvate and 2-oxoglutarate was 72±7%.

Determination of intracellular amino acid pools in G. intestinalis.
Intracellular amino acid analysis was performed as described previously (Knodler et al., 1994 ), with 3-aminopropionate as the internal standard, on a Beckman 6300 amino acid analyser.

Flow cytometry.
Cellular fluorescence (green emission, 530–540 nm) was monitored by flow cytometry using a MoFlo cytometer (Cytomation PTY) with excitation at 488 nm from a water-cooled 200 mW argon-ion laser. In addition, forward light scatter and right-angle side scatter were measured and used for gating data collection. Typically, signals from >=50000 cells were acquired and analysed using Cyclops software (Cytomation PTY) for each sample. The flow-cytometric histogram shown is representative of at least three independent experiments performed with both G. intestinalis and H. inflata.

Monitoring of intracellular reactive oxygen species production.
Dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) was used to detect the intracellular generation of reactive oxygen intermediates (predominantly H2O2). H2DCFDA was added (final concentration 2 µM) to suspensions of G. intestinalis and H. inflata in PBS (approx. 5x106 cells ml-1) and fluorescence was measured by flow cytometry or on-line using a Perkin Elmer LS 50B luminescence spectrophotometer (excitation 504 nm, emission 527 nm). H2DCFDA is non-fluorescent and is able to permeate biological membranes. Once in the cytosol, esterase activity renders the indicator non-permeant by forming the fluorescent product dichlorofluorescein, and the fluorescence intensity of the dye is proportional to the rate of oxidation by reactive oxygen species (predominantly H2O2). The fluorescence signals detected from H. inflata H2DCFDA-loaded cells were larger than those from G. intestinalis and S. cerevisiae cells. These differences are believed to reflect dye-loading efficiencies and do not compromise the assays. The fluorescence traces shown are representative of at least three independent experiments performed with both G. intestinalis and H. inflata.

Measurement of photo-emissive O2-reduction products in H. inflata.
H. inflata cells in PBS (approx. 6·5x106 cells ml-1) were exposed to low levels of dissolved O2 in a continuously stirred reactor (200 r.p.m.) fitted with an observation window for photon counting (Lloyd et al., 1979 , 1985 ). The reactor was open for gas exchange from a mobile gas phase, the composition of which was controlled using a gas mixer; dissolved O2 was monitored using a Radiometer electrode. A Peltier-cooled, red-sensitive, photon-counting photo-multiplier device (EMI 9817) provided continuous monitoring of weak chemiluminescence emission (>900 nm) without spectral discrimination. The O2 concentration of air-saturated water at 25 °C was taken to be 253 µM O2 (Wilhelm et al., 1977 ). Data are representative of three experiments.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Initially, the study was concerned with measuring the physiological level of pyruvate and other 2-oxo acids in G. intestinalis. As described in Methods, 2-oxo acids were pre-purified from the cell extracts using hydrazide gel, and were derivatized with o-phenylenediamine, producing the 2-quinoxalinol derivatives. The pre-purification of 2-oxo acids was essential, since without this step, no discrete separation was observed. The intracellular 2-oxo acid pool concentrations in G. intestinalis together with those of their amino acid counterparts (for comparison) are given in Table 1. The number of intracellular 2-oxo acids was smaller, and their concentrations lower, than those of their amino acid counterparts, only seven 2-oxo acids versus 25 amino acids being detected (not all shown). The major 2-oxo acid detected was pyruvate (0·5 mM), which is consistent with its amino form, alanine, being present at the highest intracellular concentration. As H. inflata has a similar intracellular concentration of amino acids to that of G. intestinalis (Biagini et al., 2000 ), it was therefore assumed that their pyruvate levels would also be comparable. Experiments were then conducted to test whether pyruvate, added at concentrations close to physiological levels, would confer protection against reactive oxygen species.


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Table 1. Intracellular 2-oxo acids and their amino forms in G. intestinalis

 
We have previously observed that incubation of diplomonads in air-saturated solutions results in the generation of reactive oxygen species (e.g. H2O2) (Biagini et al., 1997 ; Lloyd et al., 2000 ). An H2DCFDA-based assay was designed (see Methods) to monitor the intracellular generation of reactive oxygen species on-line.

Flow cytometry was used to confirm that fluorescence of H2DCFDA-loaded G. intestinalis and H. inflata cells was arising intracellularly. Analysis of unstained H. inflata cells in PBS revealed a small degree of autofluorescence, whereas H2DCFDA-loaded cells (15 min incubation) were observed to have a significantly increased intracellular fluorescence intensity (Fig. 1). Addition of H2O2 (200 µM) to H2DCFDA-loaded cells further increased intracellular fluorescence, indicating free diffusion of H2O2 into the cell cytosol. Prior incubation of H2DCFDA-loaded cells with pyruvate (1 mM, 5 min) attenuated the H2O2-induced fluorescence (Fig. 1). Similar results were also observed with G. intestinalis trophozoites (not shown).



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Fig. 1. Flow cytometric histograms of intracellular fluorescence intensity (488 nm excitation, 530–540 nm emission) in H. inflata trophozoites. The various cell populations reflect different treatments: (a) intrinsic autofluorescence, (b) fluorescence arising from H2DCFDA-loaded cells, (c) H2DCFDA-loaded cells in the presence of 200 µM H2O2 and (d) H2DCFDA-loaded cells, previously incubated with pyruvate (1 mM), in the presence of 200 µM H2O2.

 
The scavenging activity of pyruvate was monitored in real time with a luminescence spectrophotometer. Fluorescence from H2DCFDA-loaded H. inflata cells was monitored after the addition of 200 µM H2O2 (Fig. 2). Pyruvate was shown to reduce the rate of reactive oxygen species generation (as indicated by the rate of increase in fluorescence intensity), the degree of attenuation being proportional to the pyruvate concentration (Fig. 2). Heat-fixed (60 °C, 15 min) H2DCFDA-loaded cells were used as a negative control. A smaller but significant decrease in the rate of H2O2-induced reactive oxygen species generation by pyruvate was also observed in G. intestinalis (Table 2). Addition of pyruvate metabolism end-products such as ethanol, alanine and acetate (up to 3 mM) was shown not to decrease the rate of H2O2-induced reactive oxygen species generation in H. inflata or G. intestinalis.



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Fig. 2. Fluorimetric traces of H. inflata H2DCFDA-loaded cells. Increases in fluorescence are representative of increases in the rate of oxidative species generated (see Methods). Fluorescence was monitored from suspensions of live and heat-fixed cells (60 °C, 15 min) after the addition of H2O2 (200 µM) in the absence and presence of various concentrations of pyruvate.

 

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Table 2. Effect of pyruvate on H2O2- and menadione ()-induced generation of oxygen species in G. intestinalis and H. inflata, as measured by intracellular H2DCFDA fluorescence

 
Menadione was also used to induce the generation of intracellular reactive oxygen species (predominantly ). Fluorescence arising from H2DCFDA-loaded G. intestinalis cells was shown to increase upon the addition of 300 µM menadione (Fig. 3). The increase in the rate of fluorescence intensity (and thus the increase in the rate of reactive oxygen species generation) with menadione was shown to be reduced by the addition of 1 mM pyruvate (Fig. 3). Heat-fixed (60 °C, 15 min) H2DCFDA-loaded cells were used as a negative control. Duplication of the experiment using H. inflata cells also resulted in pyruvate (1 mM) reducing the rate of menadione-induced reactive oxygen species generation, but to a lesser extent than that shown for G. intestinalis (Table 2).



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Fig. 3. Fluorimetric traces of G. intestinalis H2DCFDA-loaded cells. Fluorescence intensity was monitored from suspensions of live and heat-fixed cells (60 °C, 15 min) after the addition of menadione (300 µM) in the absence and presence of pyruvate (1 mM).

 
Low-level chemiluminescence arises from cells predominantly as a result of singlet O2 generation (e.g. during lipid peroxidation). As such, measurement of low-level chemiluminescence is a useful assay for the monitoring of oxidative stress. Low-level chemiluminescence was observed upon oxygenation of an anaerobic suspension of H. inflata cells (Fig. 4). Prior anaerobic incubation of trophozoites with pyruvate (1 mM) dramatically lowered the burst of chemiluminescence occurring upon oxygenation of the suspending solution (Fig. 5).



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Fig. 4. Chemiluminescence of a whole-cell suspension of H. inflata. Low-level chemiluminescence was monitored in a continuously stirred reactor, open for gas exchange. Traces indicate the response of an anaerobic suspension of H. inflata in PBS to oxygenation. (a) [O2], (b) chemiluminescence.

 


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Fig. 5. Chemiluminescence of a whole-cell suspension of H. inflata. Traces indicate the response of an anaerobic suspension of H. inflata in PBS to oxygenation in the presence of pyruvate (1 mM). (a) [O2], (b) chemiluminescence.

 
One of the hypotheses leading to this study was that the observed reactive oxygen species scavenging activity of pyruvate favoured anaerobic energy metabolism over oxidative metabolism. To test this hypothesis, S. cerevisiae cells (which had been grown aerobically) in PBS were loaded with H2DCFDA and the subsequent fluorescence was monitored continuously with the luminescence spectrophotometer. The fluorescence arising from the H2DCFDA-loaded yeast cells was shown to increase proportionally with pyruvate concentration (Fig. 6a). In addition, the rate of increase in fluorescence intensity in H2DCFDA-loaded yeast cells, oxidatively stressed by the addition of H2O2 (200 µM), was not attenuated by the addition of up to 3 mM pyruvate (Fig. 6b). These data indicate that, under our in vitro conditions, pyruvate does not have a net antioxidant activity in aerobically grown yeast, but rather is acting as a generator of reactive oxygen species.



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Fig. 6. Fluorimetric traces of S. cerevisiae H2DCFDA-loaded cells. (a) Fluorescence intensity was monitored from a cell suspension of S. cerevisiae in PBS during the addition of various concentrations of pyruvate. (b) Fluorescence intensity was monitored from a cell suspension of S. cerevisiae in PBS after the addition of H2O2 (200 µM) and pyruvate (3 mM).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we have measured the intracellular levels of 2-oxo acids, including pyruvate, in G. intestinalis. The ability of pyruvate, added at concentrations close to physiological levels, to act as an oxidative species ‘scavenger’ was then investigated in both G. intestinalis and H. inflata. The effect of pyruvate on the generation of reactive oxygen species in the fermentative diplomonads was then compared with that in respiring S. cerevisiae.

As shown in Table 1, the 2-oxo acid pools in G. intestinalis were shown to be smaller in number and at lower concentrations than their amino forms. Values for the intracellular amino acids were very similar to those reported previously (Knodler et al., 1994 ). The ratio of the pyruvate pool to the alanine pool was almost identical to that of the 2-oxoglutarate pool to the glutamate pool, suggesting that the reaction catalysed by alanine aminotransferase is approximately at equilibrium in this parasite. In addition to alanine, the other neutral amino acids glycine, valine, leucine and isoleucine that contribute significantly to the total amino acid pool were represented by the respective oxo-acids glyoxylate, 2-oxoisovalerate, 2-oxoisocaproate and 2-oxocaproate in the oxo acid pool. However, no oxoaloacetate pool was detected, although G. intestinalis contains aspartate aminotransferase activity (Edwards et al., 1994 ). The lack of detectable oxaloacetate may be due to its instability; alternatively, it may simply be present at undetectable levels because of the high activity of malate dehydrogenase, favouring oxaloacetate reduction. In addition, the 2-oxo acid forms of the aromatic acids phenylalanine and tyrosine were undetectable. This was also unexpected in view of the aromatic aminotransferase activities that have been previously observed in G. intestinalis (Edwards et al., 1994 ).

Biological membranes are readily permeable to pyruvate and it can therefore be assumed that the extracellular addition of pyruvate would raise, at least transiently, the levels of intracellular pyruvate. With the aid of the H2DCFDA-based assays, intracellular generation of reactive oxygen species by G. intestinalis and H. inflata was monitored in real time. As expected, the addition of H2O2 (which is uncharged and therefore freely penetrates the plasma membrane) was shown to increase the rate of intracellular generation of reactive oxygen species (as indicated by the increase in fluorescence intensity; Fig. 2, Table 2). Similarly, the addition of the synthetic quinone menadione increased the rate of intracellular generation of reactive oxygen species (Fig. 3, Table 2). Both H2O2- and menadione-induced generation of reactive oxygen species in the diplomonads were reduced by the addition of pyruvate. Pyruvate was shown to be more effective at reducing menadione-induced generation of reactive oxygen species (predominantly ) in G. intestinalis than in H. inflata (Table 2). Conversely, pyruvate was shown to be more effective at reducing H2O2-induced generation of reactive oxygen species in H. inflata than in G. intestinalis (Table 2). This disparity could be explained by the different antioxidant systems present in these organisms, e.g. H. inflata contains an Fe-type SOD (Biagini et al., 1997 ), whereas SOD is undetectable in G. intestinalis (Brown et al., 1995 ).

Pyruvate was also shown to reduce the generation of weak chemiluminescence arising from the oxygenation of the suspending solution (Fig. 4). These data signify that pyruvate, in vivo, may play a significant role as a low-molecular-mass antioxidant in G. intestinalis and H. inflata, protecting the cells from both the generation and propagation (as indicated by the reduction of singlet O2 production) of reactive oxygen species. It is also possible that the other 2-oxo acids detected, such as 2-oxoglutarate (which has known H2O2-scavenging activity; Halliwell & Gutteridge, 1999 ), may also act as physiological antioxidants.

In contrast to the effect of pyruvate in diplomonads, the addition of pyruvate to yeast had no observable antioxidant effect, but rather caused an increase in the rate of generation of reactive oxygen species (Fig. 6). The oxidative respiratory chain is a major source of H2O2 and other oxygen radicals (e.g. ) due to ‘leaky’ redox reactions (e.g. Halliwell & Gutteridge, 1999 ). It is therefore probable that pyruvate in respiring yeast cells acts to increase the generation of reactive oxygen species by increasing the flux of electrons down the respiratory chain.

In a recent elegant study performed by Brand & Hermfisse (1997) , the phenomenon of aerobic glycolysis was investigated in mitogen-activated rat thymocytes. Resting thymocytes were observed to meet their ATP demand largely by oxidative glucose catabolism, whereas the energy demand of proliferating thymocytes was satisfied by glycolysis. Decreased reactive oxygen species generation, due to the shut-down of respiration as well as an observed increase in the pyruvate pool during glycolytic metabolism, were suggested as metabolic strategies employed by the cell to minimize oxidative stress during cell division. It appears, therefore, that pyruvate can act to induce or to scavenge reactive oxygen species, depending on the metabolic mode (e.g. oxidative or glycolytic) of the cell. In diplomonads, where components of the respiratory chain are undetectable (e.g. cytochromes), the fermentative energy metabolism results in pyruvate acting as an efficient scavenger of reactive oxygen species. Whether these two diplomonads have the ability to regulate pyruvate levels in response to oxidative stress remains to be determined. It is conceivable, however, that previously described changes in metabolic end-product formation (Paget et al., 1993a ; Biagini et al., 1998 ) may indirectly promote a rise in the intracellular pyruvate availability in response to moderate increases in O2 tension.


   ACKNOWLEDGEMENTS
 
This work was supported by the Australian Research Council and by The Royal Society (D.L.).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 30 March 2001; revised 1 June 2001; accepted 11 July 2001.