Pyruvate reduces DNA damage during hypoxia and after reoxygenation in hepatocellular carcinoma cells Emilie Roudier*, Christine Bachelet and Anne Perrin ´ ´ ´ ` Unite de Biophysique Cellulaire et Moleculaire, IFR ‘RMN biomedicale: de la cellule a l’homme’, CRSSA, BP 87, La Tronche, France Keywords DNA damage; glutathione; hypoxia; pyruvate; reoxygenation Correspondence ´ A Perrin, CRSSA ⁄ RBP, Unite de ´ biophysique cellulaire et moleculaire, 24 ´ Avenue des Maquis du Gresivaudan, BP 87, 38702 La Tronche Cedex, France Fax ⁄ Tel: + 33 76 63 68 79 E-mail: aperrin@crssa.net *Present address ´ ´ ´ Departement de kinesiologie, Universite de ´ Montreal, Canada (Received July 2007, revised 10 August 2007, accepted 14 August 2007) doi:10.1111/j.1742-4658.2007.06044.x Pyruvate is located at a crucial crossroad of cellular metabolism between the aerobic and anaerobic pathways Modulation of the fate of pyruvate, in one direction or another, can be important for adaptative response to hypoxia followed by reoxygenation This could alter functioning of the antioxidant system and have protective effects against DNA damage induced by such stress Transient hypoxia and alterations of pyruvate metabolism are observed in tumors This could be advantageous for cancer cells in such stressful conditions However, the effect of pyruvate in tumor cells is poorly documented during hypoxia ⁄ reoxygenation In this study, we showed that cells had a greater need for pyruvate during hypoxia Pyruvate decreased the number of DNA breaks, and might favor DNA repair We demonstrated that pyruvate was a precursor for the biosynthesis of glutathione through oxidative metabolism in HepG2 cells Therefore, glutathione decreased during hypoxia, but was restored after reoxygenation Pyruvate had beneficial effects on glutathione depletion and DNA breaks induced after reoxygenation Our results provide more evidence that the a-keto acid promotes the adaptive response to hypoxia followed by reoxygenation Pyruvate might thus help to protect cancer cells under such stressful conditions, which might be harmful for patients with tumors Pyruvate, as well as lactate, is an end-product of glycolysis Its production is enhanced in tumor cells, where high rates of aerobic glycolysis, historically known as the Warburg effect, are observed [1] It is only lately that pyruvate has been described as playing an important role in cancer progression First of all, alterations in components of pyruvate metabolism have been reported in tumor cells, and appeared to increase cancer cell proliferation [2,3] Moreover, recent evidence supports a novel role of pyruvate in metabolic signaling in tumors Pyruvate has been reported to promote hypoxia-inducible factor (HIF-1) stability and activate HIF-1-inducible gene expression This can promote the malignant transformation and survival of cancer cells [4,5] Pyruvate also exhibits strong angiogenic activity in vitro and in vivo and positively affects angiogenic processes [6] As the angiogenic switch is a crucial event in tumorigenesis, pyruvate may be important for cancer progression All together, these findings suggest that pyruvate could induce the molecular signaling usually caused by hypoxia Chronic or transient hypoxia in the tumor is induced by heterogeneous bloodflow resulting from impaired vascularization [7] Tumor cells are often exposed to shorter or longer periods of hypoxia or ischemia followed by reoxygenation or recirculation An adaptive response of cancer cells takes place through multifaceted changes [8], which are mainly coordinated by HIF-1 [9] The outcome is clonal selection of the tumor cells that are most resistant and well adapted to hypoxia [10] Many alterations occur that induce Abbreviations GSH(c-glutamyl), c-glutamyl glutathione; HIF-1, hypoxia-inducible factor; PCA, perchloric acid; ROS, reactive oxygen species 5188 FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works Pyruvate reduces DNA damage during ⁄ after hypoxia E Roudier et al promote adaptative responses to hypoxia followed by reoxygenation Pyruvate might thus help to protect of cancer cells during such stressful conditions, which might be harmful for patients with tumors Results HepG2 cells have greater pyruvate requirements under hypoxic conditions Pyruvate content was quantified in cell culture medium under normoxic and hypoxic conditions, and in the absence and presence of pyruvate (0.8 mm) in the culture medium (Fig 1) Total oxygen depletion was complete after h of incubation in the anaerobic jar (see Experimental procedures) The detection limits of the assay did not allow measurement of intracellular pyruvate Under normoxic conditions and in the absence of pyruvate, HepG2 cells produced and secreted pyruvate The extracellular level rose linearly up to 2.2 lmolỈ mg)1 protein after h, corresponding to 0.4 mm in the culture medium When added to the culture medium, the cells consumed pyruvate, with its concentration decreasing slightly and linearly from 0.75 mm to 0.63 mm between and h of incubation Under hypoxic conditions, and in the absence of exogenous pyruvate, pyruvate excretion by the cells remained identical to that seen under normoxia for the first hour and slowed down during the second hour After 1–2 h of incubation (depending on the experiments, data not shown), HepG2 cells stopped secreting pyruvate and consumed it, as shown by the decrease in pyruvate concentration After reoxygenation (data not shown), the production of pyruvate started again until 0.9 Extracellular pyruvate (mM) deleterious effects, such as resistance to anticancer treatment, tumor growth, and metastasis development [11,12] Moreover, they allow survival under low partial pressure of oxygen One of the alterations induced by hypoxia involves modifications of glutathione metabolism Glutathione is an important intracellular antioxidant and redox potential regulator that plays a vital role in drug detoxification and in cellular protection against damage by free radicals, peroxides, and toxins [13] Hypoxia enhances the expression of c-glutamylcysteine synthetase [14] and glutathione transferase [15] in cancer cells Such alterations of the glutathione system can enhance survival of cancer cells In general, hypoxia and reoxygenation may induce important modifications to the functioning of the antioxidative system Multilevel adaptation to oxidative damage could then follow, tending to enhance protective mechanisms This could have beneficial effects on protection against DNA damage [16] Pyruvate may play a role in this adaptive response This has been intensively studied in normal cells and tissues, such as heart, liver, and brain Generally, this a-keto acid is associated with protective effects against hypoxia and reoxygenation This is mainly ascribed to its ability to maintain redox status [17], intervening in the DNA repair system [18–20] and restoring antioxidant capacities [21–26] This adaptive response, beneficial in the case of normal cells and tissues, could become deleterious for tumor carriers when it takes place in malignant cells [27,28] However, the role of pyruvate during hypoxia is poorly documented in cancer cells We previously showed that pyruvate could favor the glycolytic pathway from glucose to lactate in glial and hepatic cells underhypoxic conditions [29] We now investigated whether such an effect might affect the adaptive response of tumor cells to hypoxia and reoxygenation We particularly focused on the antioxidant system, in particular glutathione metabolism, and on studying DNA damage The metabolism of glutathione and DNA breaks were investigated in hepatocellular carcinoma HepG2 cells cultivated with or without pyruvate during and after hypoxia In the present study, we showed that cells had a greater need for pyruvate during hypoxia Pyruvate decreased DNA breaks and might favor DNA repair We demonstrated that pyruvate was a precursor for the biosynthesis of glutathione through oxidative metabolism in HepG2 cells Therefore, glutathione decreased during hypoxia, but was restored after reoxygenation Pyruvate had beneficial effects on glutathione depletion and DNA breaks induced after hypoxia Our results provide more evidence that a-keto acids 0.8 0.7 0.6 Normoxia without pyruvate 0.5 Normoxia with pyruvate 0.4 Hypoxia without pyruvate Hypoxia with pyruvate 0.3 0.2 0.1 0.0 Time (hours) Fig Extracellular pyruvate content in culture medium of HepG2 cells under normoxia (in dark) and hypoxia (in white) when exogenous pyruvate (0.8 mM) is present (circles) or not present (square) The level of extracellular pyruvate was quantified every hour Values are means ± SD of three independent experiments FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works 5189 Pyruvate reduces DNA damage during ⁄ after hypoxia E Roudier et al Pyruvate decreases oxidative stress during hydrogen peroxide exposure Antioxidative properties have been widely ascribed to pyruvate We therefore investigated whether pyruvate might have those antioxidant capacities in our experimental conditions HepG2 cells were cultivated in medium enriched with 5.5 mm glucose in the absence and presence of pyruvate (0.8 mm) Cells were exposed to increasing doses of hydrogen peroxide from 25 to 300 lm Figure shows the level of reactive oxygen species (ROS) in HepG2 cells with and without pyruvate The ROS level was lowered in the presence of pyruvate for the range of concentrations from 75 to 300 lm hydrogen peroxide At 240 lm exposure and after normalization to a control without any cells (100%), the levels of ROS were 71% and 54% for cells incubated without and with pyruvate, respectively In our experimental conditions, pyruvate could thus decrease the generation of oxidative stress induced by Radical oxygen species (fluorescence units / s) 12000 without pyruvate 10000 with pyruvate 8000 hydrogen peroxide in HepG2 cells and may also have played a direct antioxidant role in HepG2 cells Exogenous pyruvate protects DNA under hypoxia and after reoxygenation We wondered whether the increased pyruvate uptake might be related to a protective effect against the consequences of hypoxic stress As both hypoxia and reoxygenation have been reported to induce DNA damage [30–32], we examined the effects of pyruvate addition during hypoxia and after reoxygenation on DNA damage HepG2 cells were incubated for h under normoxic and hypoxic conditions without and with pyruvate (0.8 mm) DNA fragmentation was estimated with the comet assay The assay was carried out immediately after the h incubation period, and then and h after reoxygenation of the cell cultures (Fig 3) Under normoxia, DNA fragmentation remained unchanged irrespective of the condition (with or without pyruvate) or incubation time After h under hypoxia and in the absence of pyruvate, an increase in DNA fragmentation was observed This increase was not observed in the presence of pyruvate One hour after reoxygenation, DNA fragmentation reached a maximum in both the absence and the presence of pyruvate, and thereafter decreased The increase induced by h of reoxygenation was lower in the presence of 0.8 mm pyruvate, although 40 35 DNA fragmentation (Tail extent moment) a level of approximately 0.4 mm was reached When added to the culture medium, exogenous pyruvate disappeared linearly at a higher rate than under normoxia up to h, and then more rapidly from to h of incubation The concentration stabilized at 0.4 mm The cells seemed to adjust between production and consumption of pyruvate in order to maintain the extracellular pyruvate level at about 0.4 mm in the culture medium This level was maintained after h (data not shown) This could not be achieved under hypoxia when no exogenous pyruvate was added Moreover, these data show that HepG2 cells have a higher uptake of pyruvate under hypoxic conditions 6h h + h reoxygenation 30 h + h reoxygenation * * 25 * † 20 † 15 10 6000 4000 Normoxia without pyruvate 2000 Normoxia with pyruvate Hypoxia without pyruvate Hypoxia with pyruvate Conditions 0 50 100 150 200 250 300 350 H2O2(µM) Fig Pyruvate decreases the level of ROS in the medium of HepG2 cells exposed to hydrogen peroxide HepG2 cells were incubated with 5.5 mM glucose in the presence of 0.8 mM pyruvate, and exposed to increasing doses of hydrogen peroxide (from to 300 lM) Levels of ROS were estimated by spectrofluorometry using 2¢,7¢-dichlorodihydrofluoresceine bi-acetate The data are from one representative experiment 5190 Fig Effect of pyruvate on DNA fragmentation induced by hypoxia and reoxygenation in HepG2 cells Cells were incubated for h with glucose (5.5 mM) in the absence and presence of pyruvate (0.8 mM) under normoxic or hypoxic conditions Thereafter, cells exposed to hypoxia were incubated under normoxic conditions (reoxygenation) for or h Other conditions and statistical calculations are described under Experimental procedures Values are means ± SD, and P-values ¼ 0.05 are compared in the following way: *hypoxia versus normoxia;  with versus without 13 pyruvate FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works Pyruvate reduces DNA damage during ⁄ after hypoxia nonsignificant After h of reoxygenation, DNA damage returned to normoxic levels in the cells treated with mm pyruvate, whereas it remained higher in nontreated cells (47%) These results show that the presence of pyruvate significantly reduces DNA fragmentation induced by hypoxia and reoxygenation Moreover, the comet assay has recently been described as an efficient method for detecting DNA repair [33] We therefore concluded that, after reoxygenation, HepG2 cells cultivated with pyruvate repair DNA damage sooner Intracellular glutathione (reduced and oxidized nmol/mg of protein) E Roudier et al 160 6h 140 h + h reoxygenation 120 h + h reoxygenation † 100 * 80 * * 60 * 40 20 normoxia without pyruvate normoxia with pyruvate hypoxia without pyruvate hypoxia with pyruvate Conditions Exogenous pyruvate restores glutathione levels after reoxygenation but is ineffective at maintaining glutathione levels during hypoxia Glutathione is one of the main antioxidant compounds in the cell, and it is also essential for DNA synthesis and repair [34] We wondered whether the beneficial effect of pyruvate on DNA damage could be mediated by glutathione To answer this question, we analyzed the effect of pyruvate on the glutathione content of HepG2 cells during hypoxia and after reoxygenation HepG2 cells were incubated for h under normoxic and hypoxic conditions in the absence and presence of pyruvate (0.8 mm) Thereafter, hypoxic cells were reincubated under normoxic conditions (reoxygenation) The total intracellular glutathione content (oxidized and reduced forms) was assayed in the cells immediately after incubation (6 h), and and h after reoxygenation (Fig 4) Under normoxia, intracellular reduced and oxidized glutathione levels remained unchanged irrespective of the presence of pyruvate and incubation time After h under hypoxia, the glutathione content decreased by approximately 50%, independently of the presence of pyruvate This profile remained unchanged after h of reoxygenation After h of reoxygenation, a clear difference was observed between cells incubated with and without pyruvate When pyruvate was lacking, the glutathione levels remained significantly low, whereas in the presence of the a-keto acid, the glutathione level was restored Pyruvate is a precursor of glutathione under normoxia To further investigate the effects of pyruvate on glutathione content during hypoxia, we examined pyruvate metabolism using 13C-NMR The distribution of 13C labeling was analyzed after incubation of HepG2 cells in a culture medium containing [13C3]pyruvate, under normoxia and hypoxia, for h Fig Effect of pyruvate on glutathione content during hypoxia ⁄ reoxygenation in HepG2 cells Cells were incubated for h with glucose (5.5 mM) in the absence and presence of pyruvate (0.8 mM) under normoxic or hypoxic conditions Thereafter, cells exposed to hypoxia were incubated under normoxic conditions (reoxygenation) for or h Levels of oxidized and reduced glutathione were measured in the cell extracts Other conditions and statistical calculations are described under Experimental procedures Values are means ± SD, and P-values ¼ 0.05 are compared in the following way: *hypoxia versus normoxia;  with versus 14 without pyruvate [13C3]Pyruvate mainly led to the production of C-enriched lactate, alanine, glutamate, glutamine and glutathione as previously described (data not shown) Under hypoxia, the decreased abundance of 13 C-enriched glutamine and glutamate was concomitant with the increased abundance of enriched lactate and alanine This is due to the blockade of the tricarboxylic acid cycle induced by lack of oxygen We examined the peaks corresponding to the glutathione carbons, and quantified the relative abundance of the corresponding molecules under hypoxia and normoxia (Fig 5A) Incubation with [13C3]pyruvate resulted in labeling of [13C2]c-glutamyl glutathione [GSH(c-glutamyl)], [13C4]c-glutamyl glutathione and [13C3]cglutamyl glutathione Peaks corresponding to [13C3]glutamine and [13C3]GSH(c-glutamyl) were indistinguishable, as were those corresponding to [13C2]glutamine and [13C2]GSH(c-glutamyl) The presence of [13C4]GSH(c-glutamyl) indicated that HepG2 cells consumed pyruvate to form glutathione under normoxia Hypoxia induced decreases, respectively, of 92% and 82% in 13C2 and 13C3 glutamine ⁄ glutathione peak intensities The [13C4]glutathione peak was reduced by 85% This dramatic decrease indicated that hypoxia induced strong inhibition of the glutathione synthesis pathway from pyruvate We also analyzed intracellular glutathione (oxidized and reduced form) by enzymatic assay during normoxia 13 FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works 5191 Pyruvate reduces DNA damage during ⁄ after hypoxia E Roudier et al Relative peak area (arbitrary unit) A 3.5 normoxia 3.0 hypoxia 2.5 2.11 2.0 1.5 1.0 0.38 0.5 0.0 Intracellular glutathione (reduced and oxidized nmol/mg of protein) Glutathione (reduced and oxidized nmol/mg of protein) 0.31 0.20 [2- 13C]-GSH [4- 13C]-GSH [3- 13C]- GSH and/or -Gln and/or -Gln 13C-enriched metabolites B C 2.52 2.07 175 150 Normoxia without pyruvate Normoxia with pyruvate 125 Hypoxia with pyruvate Hypoxia without pyruvate 100 * * * 75 * 50 Fig Effect of hypoxia and pyruvate on glutathione synthesis by HepG2 cells (A) Glutathione 13C labeling following incubation of the cells with 13C-enriched pyruvate: cells were incubated for h with [13C3]pyruvate and 5.5 mM glucose under normoxic and hypoxic conditions After NMR analysis, the peaks corresponding to [13C3]glutamine (Gln) and ⁄ or [13C3]GSH (c-glutamyl), [13C4]GSH(c-glutamyl), and [13C2]glutamine and ⁄ or [13C2]GSH(c-glutamyl) were identified The relative amount was quantified by integration of the peak area (B) Intracellular kinetics of intracellular glutathione (oxidized and reduced) concentration: biochemical measurement of total glutathione was performed every hour HepG2 cells were cultivated in the absence (squares) and presence (circles) of pyruvate under normoxic (in black) or hypoxic (in white) conditions Other conditions and statistical calculations are described under Experimental procedures Values are means ± SD; *P ¼ 0.05 as compared with corresponding normoxic condition (C) Distribution of total glutathione in the intracellular and extracellular compartments: HepG2 cells were incubated for h under normoxic and hypoxic conditions in the absence and presence of pyruvate (1 mM) Intracellular and extracellular total glutathione contents were assayed separately The sum of both is also shown Values are means ± SD; *P ¼ 0.05 as compared with the corresponding normoxic condition 25 0 175 150 125 100 75 Time (hours) Normoxia with pyruvate Normoxia with pyruvate Hypoxia without pyruvate * Hypoxia with pyruvate * * * overall decrease in the glutathione level This effect was independent of pyruvate supplementation Together, these data indicate that pyruvate is a precursor of glutathione under normoxic conditions through glutamate generated by oxidative metabolism (Fig 6) However, pyruvate cannot be used as a glutathione precursor under hypoxia, because of the lack of oxygen 50 25 * * Discussion Extracellular Intracellular Localization Extracellular and intracellular and hypoxia (from to h, Fig 5B) In both the presence and the absence of pyruvate, a regular time-dependent increase in glutathione content occurred under normoxia, whereas no variation was observed under hypoxia After h of incubation, the glutathione level in cells exposed to hypoxia was significantly lower than in cells exposed to normoxia, confirming that addition of exogenous pyruvate failed to restore glutathione synthesis To confirm that the unchanged intracellular glutathione was due to inhibition of synthesis and not simply excretion from the cell during hypoxia, we also analyzed extracellular levels after h of incubation (Fig 5C shows intracellular and extracellular glutathione levels and the sum of both) Even though the extracellular content increased in response to hypoxia, indicating release by the cells, the sum of both contents showed an 5192 Modulation of the fate of pyruvate fate in one direction or another can be important for adaptive responses to hypoxia followed by reoxygenation [26,35] Repression of pyruvate dehydrogenase (EC 1.2.4.1) and switching between the highly active tetrameric and the inactive dimeric forms of pyruvate kinase (EC 2.7.1.40) are observed in tumors [2,3] Such alterations of pyruvate metabolism could be advantageous for cancer cells under such stressful conditions [36] Our present work provides new insights into the role of pyruvate in tumor cells during hypoxia We show here that tumor HepG2 cells are inclined to maintain extracellular pyruvate at a constant level (0.4 mm) Hypoxia inhibits such regulation, whereas pyruvate supplementation restores it We also observed that hypoxic cells increase their consumption of exogenous pyruvate and stop releasing it when the a-keto acid is absent from the medium Our data indicate that the endogenous need for pyruvate increases under hypoxia Pyruvate protects cells from DNA breaks induced by both hypoxia and reoxygenation FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works Pyruvate reduces DNA damage during ⁄ after hypoxia E Roudier et al Extracellular area Glucose Amino acids (AA) Cysteinylglycine conjugate 2NAD + + 2ADP + Pi Glycolysis H+ 2NADH, + 2ATP GS-X (conjugate) γ-Glutamyl -AA Pyruvate × Cysteinylglycine ADP + Pi 2× Alanine 2× + NADH, H Glutathione GSH 2× Lactate 2×NAD + Glycine + ATP ADP + Pi 2×Pyruvate 2×NADH, H + + 2×CO2 2×NAD + γ-Glutamyl-cysteine CoA 2×Acetyl-CoA Cysteine CoA + ATP γ-Glutamyl -AA Glutamate 5-Oxoproline Glutamate 4×CO2 Krebs Cycle Glutamine 6×NAD + ×FADH, H + ×FAD + ×NADH, H + 2ATP Cytosol 2ADP + Pi Mitochondrion Fig Pathway of glutathione synthesis from pyruvate, showing how pyruvate, through the tricarboxylic acid cycle, is involved in glutamate synthesis and then in the formation of glutathione (GSH) That is not well correlated with the beneficial effect on the antioxidant glutathione Actually, pyruvate restores glutathione levels only during reoxygenation, and has no effect under hypoxia We demonstrate here that the a-keto acid is a precursor of glutathione through the tricarboxylic acid cycle, and that hypoxia severely impairs its biosynthesis Regulation of extracellular pyruvate content has previously been reported in tumor cells as well as in nontumor cells [37] This mechanism is assumed to be a way for cells to lower oxidative stress Pyruvate has antioxidant properties [21,22] that could possibly also be manifested under our experimental conditions The phenomenon described by O’Donnell-Tormey might well take place in HepG2 cells Impairment of the regulation of the extracellular content and the increase of its uptake induced by hypoxia show that alteration of pyruvate metabolism takes place The greater uptake might be due to an increased need to maintain antioxidant capacity in the cells However, it might also result from a metabolic requirement Pyruvate increases the ratio of lactate production to glucose consumption (+ 19%, data not shown) This is in line with our previous work [29] indicating enhancement of glycolysis activity in the presence of pyruvate Increased glycolysis might thus maintain the ATP supply during oxygen deprivation while oxidative phosphorylation is seriously impaired Such an increase has been reported to have protective effects against hypoxic stress [38,39] Hypoxia, known to alter trhe antioxidant system, affects glutathione metabolism as well In our experimental conditions, we observed a decrease of intracellular content together with release of the glutathione In rat primary hepatocytes in vitro, hypoxia induced activation of the glutathione transporters, resulting in increased glutathione export [40] A similar mechanism might occur in HepG2 cells, where glutathione transport is functional [41] Furthermore, our results also show that hypoxia induces a decrease in total glutathione Pyruvate has no effect on the hypoxia-induced decrease of glutathione content, whereas it allows complete restoration of total glutathione after reoxygenation Hypoxia-induced inhibition of glutathione synthesis from pyruvate might be the main mechanism responsible for such an effect Under normoxia, we demonstrate that pyruvate supplies glutamate through oxidative metabolism in tumor cells Inhibition of the FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works 5193 Pyruvate reduces DNA damage during ⁄ after hypoxia tricarboxylic acid cycle thus results in significant inhibition of glutathione synthesis Usually, cysteine is assumed to be the limiting precursor of the two-step reaction leading to glutathione synthesis However, glutamate plays an important role in glutathione synthesis [3] Negative feedback from glutathione itself on the step catalyzed by glutamate-cysteine ligase can be prevented by glutamate [1,2] It might even become limiting under conditions of mitochondrion blockage in muscle [4] and during hypoxia in glial cells [5] Pyruvate enhances glycolysis activity during hypoxia in HepG2 cells [29] By providing more substrates for the tricarboxylic acid cycle and through indirect effects on redox status, pyruvate might thus favor restoration of the glutamate pool and then the glutathione pool after reoxygenation (Fig 6) Induction of DNA damage by hypoxia and reoxygenation is a well-known phenomenon mainly caused by ROS [30–32] A functioning antioxidant system is essential to reduce such damage Despite the absence of an effect on glutathione, pyruvate has beneficial effects on DNA breaks under hypoxia Many studies have reported that pyruvate improves antioxidant capacities and protects normal tissues against damage induced by hypoxia ⁄ reoxygenation and ischemia ⁄ reperfusion [23,26] The increased uptake of pyruvate might allow HepG2 cells to maintain their antioxidative capacities in a way independent of glutathione during hypoxia However, after reoxygenation, the beneficial effect of pyruvate against DNA breaks is well correlated with glutathione restoration DNA breaks decrease faster in cells treated with pyruvate, suggesting stimulation of DNA repair As glutathione is essential for DNA synthesis in general, as well as for DNA repair [34], the action of pyruvate on glutathione might also favor DNA repair During hemorrhagic shock and ischemia followed by reperfusion, pyruvate interferes with poly(ADP-ribose)-polymerase (EC 2.4.2.30) activity by preventing loss of total NAD+ content [19,20,42] This might enhance DNA repair Pyruvate thus plays a role in DNA protection and repair in tumor HepG2 cells during hypoxia and after reoxygenation The mechanism might involve both antioxidant properties and metabolic activity The exact mechanism underlying the pyruvate effects has yet to be further examined In light of the data in the literature and our work with HepG2 cells, we concluded that pyruvate might act similarly in tumor cells and nontumor cells It could then act as a protective agent against DNA damage under conditions of tumor hypoxia Many strategies for cancer therapy are based on increases in the number of DNA strand breaks (e.g radiotherapy and alkylating agents) Pyruvate might thus reduce the efficiency 5194 E Roudier et al of treatment by limiting DNA damage and favoring repair Pyruvate also stimulates angiogenesis [4,6]; this might favor tumor reoxygenation and progression Furthermore, a high intracellular glutathione level correlates with a high level of proliferation of tumor cells [43] and with resistance to anticancer treatment [44,45] Pyruvate might support a high glutathione level in reoxygenated tumors All of these effects might favor tumor development and lower efficacy of some therapies They remain to be verified in vivo in tumors If they were verified, it would confirm that no matter how pyruvate acted, it would be deleterious in cancer In conclusion, this study confirms the importance and the multilevel and very complex implications of pyruvate for cell responses to hypoxia ⁄ reoxygenation Our results are in agreement with current literature identifying pyruvate as a protector of normal and tumor cells subjected to hypoxia Furthermore, this study provides additional data confirming that whatever the underlying mechanisms at work, the presence of pyruvate is undesirable in tumor cells Indeed, controlling pyruvate levels might be an advantageous way of modulating tumor resistance and improving the efficiency of certain cancer therapies Experimental procedures Cell culture A human hepatocellular carcinoma HepG2 cell line was purchased from the American Type Cell Collection It derives originally from a hepatocellular carcinoma biopsy and synthesizes nearly all human plasma proteins [46] The cell line is not tumorigenic in immunosuppressed mice Cells, used between passages 76 and 82, were grown in Petri dishes coated with type collagen (10 lgỈmL)1, 60 lLỈcm)2, 30 at 37 °C; Sigma-Aldrich, Saint-Quentin Fallavier, France) in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Biowhittaker, Cambrex Corporate, East Rutherford, NJ), antibiotics (100 000 L)1 penicillin, 100 mgỈL)1 streptomycin; Boehringer Ingelheim, Paris, France), mm glutamine (Jacques Boy Institute, Reims, France), 1% nonessential amino acids and mm pyruvate (Seromed Biochrom KG, Berlin, Germany) in a 95% air ⁄ 5% CO2 humidified atmosphere Half of the medium was changed every days The cell cultures were split at confluence with 0.25% (w ⁄ v) trypsin (Jacques Boy Institute) and seeded at a density of · 104 cellsỈcm)2 For the experiments, cells were used at subconfluency (2 days after passage) as determined by the growth curve based on cell numbers and protein quantification To rule out the presence of mycoplasma contamination, tests were performed using a commercially available detection kit (Polylabo ⁄ VWR International, Fontenay- FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works E Roudier et al sous-Bois, France) Cell viability was routinely determined using Trypan Blue exclusion Incubation of cells Cells were incubated as previously described [29] Briefly, subconfluent HepG2 cells were incubated for h with DMEM base (Sigma-Aldrich) containing 5.5 mm glucose, 10 mm Hepes (Sigma-Aldrich), 10% fetal bovine serum, antibiotics (100 000 L)1 penicillin, 100 mgỈL)1 streptomycin), 1% nonessential amino acids and mm glutamine, both without and with pyruvate (1% of a 100 mm stock solution, 0.8 mm final), in a normoxic atmosphere with 5% CO2 and under low oxygen pressure in anaerobic jars The oxygen content in the jars was monitored with an oxygen electrode (O2 sensor; Mettler-Toledo, Viroflay, France) Similar results were obtained by incubating the cells in an oxygen-depleted atmosphere using a cell culture incubator with N2, O2 and CO2 control (Queue Systems Inc., Asheville, NC) For the reoxygenation experiments, cells incubated under hypoxia were removed from the jar and placed in a normoxic atmosphere with 5% CO2 Extraction and quantification of extracellular pyruvate Perchloric acid (PCA) (8%, v ⁄ v) was added to the culture medium (2 : 1, v ⁄ v) After homogenization, the mixture was centrifuged (4000 g, 10 min, °C, CR3i centrifuge and swing-out rotor; Jouan, Saint-Herblain, France) and the supernatant was stored at ) 80 °C prior to assay Pyruvate was quantified by the method previously described by Marbach & Weil [47], with slight modifications Briefly, the assay was carried out in a 96-well plate with 160 lL of sample (PCA extract), 40 lL of Tris ⁄ HCl buffer (1.5 m Trizma base with 0.05% w ⁄ v sodium azide, pH 10.5; Sigma-Aldrich) and 40 lL of a mixture (0.3 : 2, v ⁄ v) of NADH-Na2 (4.55 mgỈmL)1) and Trizma base buffer per well First, the absorbance was measured at 340 nm: 10 lL of lactate dehydrogenase (EC 1.1.1.27) (400 mL)1 in 3.2 m ammonium sulfate, pH 6.5) was added to each well, and the absorbance was measured after complete stabilization The absorbance at 340 nm resulting from the oxidation of NADH to NAD reflected the amount of pyruvate originally present in the sample The pyruvate sample concentration was determined according to a standard curve established between and 0.5 mm pyruvate Comet assay The comet assay was performed according to the method described by Singh et al [48], using alkaline electrophoresis, which allows detection of single-strand and double-strand breaks Pyruvate reduces DNA damage during ⁄ after hypoxia Cells cultivated in Petri dishes (25 mm in diameter) were suspended in 0.5 mL of NaCl ⁄ Pi, and cell density was estimated with a Malassez slide An aliquot of the suspension was added to low molecular weight agarose (0.8%, p ⁄ v in NaCl ⁄ Pi) to obtain a final concentration of 25 · 104 cellsỈmL)1 Eighty microliters of agarose-suspended cells was placed on an agarose-coated slide (high molecular weight, 1% p ⁄ v in NaCl ⁄ Pi) and covered with a coverslip After on ice, the coverslip was gently removed The slides were incubated (1 h, °C in the dark) in lysis buffer (45 mL of buffer containing 2.5 m NaCl, 0.1 m EDTA, 10 mm Tris with mL of dimethylsulfoxide and 0.5 mL of Triton X-100, pH 10) The slides were rinsed twice with electrophoresis buffer (300 mm NaOH, mm EDTA) for 10 min, and then for 25 Electrophoresis was carried out at 25 V and 300 mA for 37 at room temperature Finally, the slides were washed with Tris ⁄ HCl buffer (pH 7.5) DNA was stained using an ethidium bromide solution (0.1 mgặmL)1, 50 lL per slide, kex ẳ 525 nm and kem ¼ 650 nm) The slides were read with an epifluorescence microscope equipped with a CDD camera (Zeiss Axioskop20, Carl Zeiss, Microscope Division, Oberkochen, Germany) The images were analyzed with the Komet 3.0 image analysis system (formerly by Kinetic Imaging; now Andor Technology, Belfast, UK) Fragmentation was expressed in Tail Extent Moment, taking into account tail length and the percentage of DNA in the comet tail Images of 500 randomly selected cells were analyzed from each sample Extraction and quantification of glutathione After incubation, 0.5 mL of water was added to the Petri dishes (100 mm in diameter) At a low temperature, the cell monolayer was removed by scraping Cells were collected in a mL tube, and the volume was adjusted to mL before addition of 0.2 mL of metaphosphoric acid (6%, p ⁄ v) After shaking (30 s), the mixture was centrifuged (4000 g, 10 min, °C, CR3i centrifuge and swing-out rotor) The pellet was used for protein quantification by the Folin– Lowry method [49] The supernatant was kept at ) 80 °C prior to glutathione assay In the case of medium samples, 0.2 mL of metaphosphoric acid (6%, p ⁄ v) was added to mL of culture medium and treated as described above Total glutathione (oxidized and reduced forms) was quantified as previously described by Tietze [50] The assay was performed in a 96-well plate Twenty microliters of sample was placed in each well with 150 lL of Mops ⁄ EDTA buffer containing 0.165 UIỈmL)1 glutathione reductase Then, 0.267 mgặmL)1 b-NADPH and 75 lL of 5,5Â-dithiobis(2-nitrobenzoic acid) solution (0.04 mg mL)1 in 0.4 m Mops ⁄ mm EDTA, pH 6.75) were added successively After shaking, the absorbance was measured at FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works 5195 Pyruvate reduces DNA damage during ⁄ after hypoxia 412 nm using a plate reader spectrophotometer (Thermo Clinical Labsystems France, Cergy Pontoise, France) A standard curve was obtained with a commercial glutathione solution (20 mm, Sigma-Aldrich) Standard samples were treated for extraction using the protocol described for biological samples PCA extraction and extracts 13 C-NMR analysis of cell For 13C-NMR, the cell line was incubated in the presence of 5.5 mm glucose (unenriched) with mm [13C3]pyruvate (Euriso-Top, Saint Aubin, France) Cells were exposed to both normoxic and hypoxic conditions After h of incubation, the medium was discarded and the cultures were washed twice with cold NaCl ⁄ Pi, immediately frozen in liquid nitrogen, and stored at ) 80 °C until further treatment PCA extraction was performed following standard procedures Briefly, 0.3 mL of 12% PCA was added to the Petri dishes (100 mm in diameter), the cell monolayer was removed by scraping with a spatula, and the cell suspensions obtained from seven individual Petri dishes were pooled After homogenization, the final cell suspension was centrifuged at 8000 g for 10 (CR3i centrifuge and swing-out rotor), and the supernatant was adjusted to 10 pH 7.4 with KOH The samples were again centrifuged at 8000 g for 10 (CR3i centrifuge and swing-out rotor), the supernatant was lyophilized, and the dry residue was dissolved in 2.2 mL of H2O ⁄ 20% D2O for NMR analysis Proton-decoupled spectra of PCA extracts were recorded 11 on a Bruker AM400 narrow-bore spectrometer equipped with a 10 mm 31P ⁄ 13C probe (Bruker, Wissembourg, France) 13C-NMR spectra were recorded at 100.62 MHz, and each spectrum was the sum of 15 000 free induction decays A 90° pulse was applied, with a repetition time of s and an acquisition time of 0.819 s The temperature was maintained at 20 °C, and the 13C chemical shifts were referenced to the resonance of tetramethylsilane at p.p.m Peak intensities were normalized to the peak intensity of ethylene glycol, used as an internal reference Relative areas of the peaks were normalized to the area of the reference peak, arbitrarily fixed at 100 The NMR analysis of the PCA extracts was reproducibly repeated four times for both normoxic and hypoxic conditions The data shown are from a representative experiment ROS measurement The determination of intracellular oxidant production is based on the oxidation of 2¢,7¢-dichlorodihydrofluorescein (Sigma-Aldrich) to the fluorescent 2¢,7¢-dichlorofluorescein HepG2 cells were incubated in the absence and presence of pyruvate (1 mm) in DMEM base containing mm glucose in the fluorescence spectrometer After addition of 2Â,7Â-dichlorodihydrouorescein (10 lgặmL)1), uorescence 5196 E Roudier et al emission was measured continuously at 520 nm after excitation at 499 nm The value of the slope was proportional to the intracellular ROS levels Statistical analysis of results The experiments were reproducibly repeated four times Values are means ± standard deviation (n ¼ separate Petri dishes) Statistical analysis of the data was done by anova The Newman–Keuls unpaired t-test was used to determine statistical significance Acknowledgements The present study was partially funded by the Institut ´ ´ Federatif de Recherche (IFR-1) ‘Biomedical NMR: From Cell to Man’ (Grenoble) In particular, we owe thanks to Professor J.-F Le Bas References Warburg O (1956) On respiratory impairment in cancer cells Science 124, 269–270 Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC & Harris AL (2005) Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma Neoplasia 7, 1–6 Mazurek S, Boschek CB, Hugo F & Eigenbrodt E (2005) Pyruvate kinase type M2 and its role in tumor growth and spreading Semin Cancer Biol 15, 300–308 Lu H, Forbes RA & Verma A (2002) Hypoxia-inducible factor activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis J Biol Chem 277, 23111–23115 Lu H, Dalgard CL, Mohyeldin A, McFate T, Tait AS & Verma A (2005) Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1 J Biol Chem 280, 41928–41939 Lee MS, Moon EJ, Lee SW, Kim MS, Kim KW & Kim YJ (2001) Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models Cancer Res 61, 3290– 3293 Minchinton AI & Fryer KH (1996) Fluctuations in the oxygenation of experimental tumours Adv Exp Med Biol 388, 353–356 Liu L & Simon MC (2004) Regulation of transcription and translation by hypoxia Cancer Biol Ther 3, 492– 497 Semenza GL (2003) Targeting HIF-1 for cancer therapy Nat Rev Cancer 3, 721–732 10 Semenza GL (2002) HIF-1 and tumor progression: pathophysiology and therapeutics Trends Mol Med 8, S62–S67 FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works Pyruvate reduces DNA damage during ⁄ after hypoxia E Roudier et al 11 Bustamante E, Morris HP & Pedersen PL (1981) Energy metabolism of tumor cells Requirement for a form of hexokinase with a propensity for mitochondrial binding J Biol Chem 256, 8699–8704 12 Mazurek S, Eigenbrodt E, Failing K & Steinberg P (1999) Alterations in the glycolytic and glutaminolytic pathways after malignant transformation of rat liver oval cells J Cell Physiol 181, 136–146 13 Meister A & Anderson ME (1983) Glutathione Annu Rev Biochem 52, 711–760 14 O’Dwyer PJ, Yao KS, Ford P, Godwin AK & Clayton M (1994) Effects of hypoxia on detoxicating enzyme activity and expression in HT29 colon adenocarcinoma cells Cancer Res 54, 3082–3087 15 Koomagi R, Mattern J & Volm M (1999) Glucose-related protein (GRP78) and its relationship to the drugresistance proteins P170, GST-pi, LRP56 and angiogenesis in non-small cell lung carcinomas Anticancer Res 19, 4333–4336 16 Stankov K & Kovacevic Z (1999) Sensitivity of the pool of adenine nucleotides to oxidative stress and protective effect of glutamine Arch Oncol 7, 141–144 17 Bassenge E, Sommer O, Schwemmer M & Bunger R (2000) Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state Am J Physiol Heart Circ Physiol 279, H2431– H2438 18 Mongan PD, Karaian J, Van Der Schuur BM, Via DK & Sharma P (2003) Pyruvate prevents poly-ADP ribose polymerase (PARP) activation, oxidative damage, and pyruvate dehydrogenase deactivation during hemorrhagic shock in swine J Surg Res 112, 180–188 19 Sharma P, Karian J, Sharma S, Liu S & Mongan PD (2003) Pyruvate ameliorates post ischemic injury of rat astrocytes and protects them against PARP mediated cell death Brain Res 992, 104–113 20 Ying W, Alano CC, Garnier P & Swanson RA (2005) NAD+ as a metabolic link between DNA damage and cell death J Neurosci Res 79, 216–223 21 Salahudeen AK, Clark EC & Nath KA (1991) Hydrogen peroxide-induced renal injury A protective role for pyruvate in vitro and in vivo J Clin Invest 88, 1886– 1893 22 Schwartz C, Morgan RL, Way LM & Way JL (1979) Antagonism of cyanide intoxication with sodium pyruvate Toxicol Appl Pharmacol 50, 437–441 23 DeBoer LW, Bekx PA, Han L & Steinke L (1993) Pyruvate enhances recovery of rat hearts after ischemia and reperfusion by preventing free radical generation Am J Physiol 265, H1571–H1576 24 Dobsak P, Courderot-Masuyer C, Zeller M, Vergely C, Laubriet A, Assem M, Eicher JC, Teyssier JR, 12 Wolf JE, Rochette L et al (1999) Antioxidative properties of pyruvate and protection of the ischemic rat 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 heart during cardioplegia J Cardiovasc Pharmacol 34, 651–659 Kerr PM, Suleiman MS & Halestrap AP (1999) Reversal of permeability transition during recovery of hearts from ischemia and its enhancement by pyruvate Am J Physiol 276, H496–H502 Borle AB & Stanko RT (1996) Pyruvate reduces anoxic injury and free radical formation in perfused rat hepatocytes Am J Physiol 270, G535–G540 Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochem J 401, 1–11 Fruehauf JP & Meyskens FLJ (2007) Reactive oxygen species: a breath of life or death Clin Cancer Res 13, 789–794 Perrin A, Roudier E, Duborjal H, Bachelet C, RivaLavieille C, Leverve X & Massarelli R (2002) Pyruvate reverses metabolic effects produced by hypoxia in glioma and hepatoma cell cultures Biochimie 84, 1003– 1011 Englander EW, Greeley GH Jr, Wang G, Perez-Polo JR & Lee HM (1999) Hypoxia-induced mitochondrial and nuclear DNA damage in the rat brain J Neurosci Res 58, 262–269 Kamiike W, Shimizu S, Hatanaka N, Morimoto Y, Miyata M, Yoshida Y & Matsuda H (1994) Hypoxia– reoxygenation causes DNA damage in hepatocytes Transplant Proc 26, 907–909 Moller P, Loft S, Lundby C & Olsen NV (2001) Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans FASEB J 15, 1181–1186 Speit G & Hartmann A (2006) The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair Methods Mol Biol 314, 275–286 Lertratanangkoon K, Savaraj N, Scimeca JM & Thomas ML (1997) Glutathione depletion-induced thymidylate insufficiency for DNA repair synthesis Biochem Biophys Res Commun 234, 470–475 Rigobello MP & Bindoli A (1993) Effect of pyruvate on rat heart thiol status during ischemia and hypoxia followed by reperfusion Mol Cell Biochem 122, 93–100 Gatenby RA & Gillies RJ (2004) Why cancers have high aerobic glycolysis? Nat Rev Cancer 4, 891–899 O’Donnell-Tormey J, Nathan CF, Lanks K, DeBoer CJ & de la Harpe J (1987) Secretion of pyruvate An antioxidant defense of mammalian cells J Exp Med 165, 500–514 Van Rooyen J, McCarthy J & Opie LH (2002) Increased glycolysis during ischaemia mediates the protective effect of glucose and insulin in the isolated rat heart despite the presence of cardiodepressant exogenous substrates Cardiovasc J S Afr 13, 103–109 Wheeler TJ, Wiegand CB & Chien S (2005) Fructose1,6-bisphosphate enhances hypothermic preservation of FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works 5197 Pyruvate reduces DNA damage during ⁄ after hypoxia 40 41 42 43 44 cardiac myocytes J Heart Lung Transplant 24, 1378– 1384 Khan S & O’Brien PJ (1997) Rapid and specific efflux of glutathione before hepatocyte injury induced by hypoxia Biochem Biophys Res Commun 238, 320–322 Sze G, Kaplowitz N, Ookhtens M & Lu SC (1993) Bidirectional membrane transport of intact glutathione in HepG2 cells Am J Physiol 265, G1128–G1134 Mongan PD, Capacchione J, West S, Karaian J, Dubois D, Keneally R & Sharma P (2002) Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine Am J Physiol Heart Circ Physiol 283, H1634– H1644 Huang ZZ, Chen C, Zeng Z, Yang H, Oh J, Chen L & Lu SC (2001) Mechanism and significance of increased glutathione level in human hepatocellular carcinoma and liver regeneration FASEB J 15, 19–21 Bump EA, Cerce BA, al-Sarraf R, Pierce SM & Koch CJ (1992) Radioprotection of DNA in isolated nuclei by naturally occurring thiols at intermediate oxygen tension Radiat Res 132, 94–104 5198 E Roudier et al 45 Godwin AK, Meister A, O’Dwyer PJ, Huang CS, Hamilton TC & Anderson ME (1992) High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis Proc Natl Acad Sci USA 89, 3070–3074 46 Aden DP, Fogel A, Plotkin S, Damjanov I & Knowles BB (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line Nature 282, 615–616 47 Marbach EP & Weil MH (1967) Rapid enzymatic measurement of blood lactate and pyruvate Use and significance of metaphosphoric acid as a common precipitant Clin Chem 13, 314–325 48 Singh NP, McCoy MT, Tice RR & Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells Exp Cell Res 175, 184–191 49 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951) Protein measurement with the Folin phenol reagent J Biol Chem 193, 265–275 50 Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues Anal Biochem 27, 502–522 FEBS Journal 274 (2007) 5188–5198 Journal compilation ª 2007 FEBS No claim to original French government works ... hypoxia and reoxygenation have been reported to induce DNA damage [30–32], we examined the effects of pyruvate addition during hypoxia and after reoxygenation on DNA damage HepG2 cells were incubated... by Singh et al [48], using alkaline electrophoresis, which allows detection of single-strand and double-strand breaks Pyruvate reduces DNA damage during ⁄ after hypoxia Cells cultivated in Petri... new insights into the role of pyruvate in tumor cells during hypoxia We show here that tumor HepG2 cells are inclined to maintain extracellular pyruvate at a constant level (0.4 mm) Hypoxia inhibits