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
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ABSTRACT |
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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.
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INTRODUCTION |
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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; G. A. Biagini, D. M. Brown & M. R. Edwards, unpublished observation in H. inflata), and catalase and non-specific peroxidase activities are undetectable (Biagini et al., 1997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.
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METHODS |
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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 min1 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, 530540 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.
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RESULTS |
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 30 March 2001;
revised 1 June 2001;
accepted 11 July 2001.