Eur J Biochem 271, 3646–3656 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04298.x Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines Lodovica Vergani1, Maura Floreani2, Aaron Russell3, Mara Ceccon1, Eleonora Napoli4, Anna Cabrelle5, Lucia Valente2, Federica Bragantini1, Bertrand Leger3 and Federica Dabbeni-Sala2 Dipartimento di Scienze Neurologiche and 2Dipartimento di Farmacologia e Anestesiologia, Universita` di Padova, Padova, Italy; Clinique Romande de Re´adaptation SUVA Care, Sion, Switzerland; 4E.Medea Scientific Institute, Conegliano Research Centre, Conegliano, Italy; 5Dipartimento di Medicina Clinica, Universita` di Padova, c/o Istituto Veneto di Medicina Molecolare, Padova, Italy Three pairs of parental (q+) and established mitochondrial DNA depleted (q0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS) Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the q0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone q0 cells Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle q0 cell lines but not in lung q0 cells GSH peroxidase activity was four times higher in all three q0 cell lines in comparison to the parental q+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury In fact bone-derived q0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2¢,7¢-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other q+ and q0 cells, both in the presence or absence of glucose Analysis of mitochondrial and cytosolic/ iron regulatory protein-1 aconitase indicated that most ROS of bone q0 cells originate from sources other than mitochondria Cellular reactive oxygen species (ROS), such as superoxide anions (2 À ), and hydrogen peroxide (H2O2), have long been held to be harmful by-products of life in an aerobic environment ROS are potentially toxic because they are highly reactive and modify several types of cellular macromolecules Lipid, protein and DNA damage can lead to cytotoxicity and mutagenesis [1] Therefore, cells have evolved elaborate defence systems to counteract the effects of ROS These include both nonenzymatic (glutathione, pyridine nucleotides, ascorbate, retinoic acid, thioredoxin and tocopherol) and enzymatic (such as superoxide dismutases, catalase, glutathione peroxidase and peroxiredoxin) pathways, which limit the rate of oxidation and thereby protect cells from oxidative stress [1,2] Notwithstanding, evidence is emerging that ROS also act as signals or mediators in many cellular processes, such as cell proliferation, differentiation, apoptosis, and senescence [3–5] The redox environment of a cell may alter the balance between apoptosis and mitosis by affecting gene expression and enzyme activity [6] Consequently, cellular redox state is increasingly accepted as a key mediator of multiple metabolic, signalling and transcriptional pathways essential for normal function and cell survival or programmed cell death [3–6] Mitochondria are certainly the major cellular site for oxygen reduction and hence the site with the greatest potential for ROS formation An estimated 0.4–0.8% [7] to 2–4% [8] of the total oxygen consumed during electron transport is reduced not to water by cytochrome c oxidase but rather to superoxide by complexes I, and III of the respiratory chain [1,7,8] ROS production increases when respiratory flux is depressed by a high ATP/ADP ratio, high electronegativity of auto-oxidizable redox carriers in Correspondence to L Vergani, Dipartimento di Scienze Neurologiche, ` Universita di Padova, c/o Istituto Veneto di Medicina Molecolare, Via Orus 2, 35129 Padova, Italy Fax: +39 049 7923271, Tel.: +39 049 7923219, E-mail: lodovica.vergani@unipd.it Abbreviations: CS, citrate synthase; CuZnSOD, copper zinc superoxide dismutase; DCF, 2¢,7¢-dichlorofluorescin; DTT, 1,4-dithio-DLthreitol; GSH, glutathione; GSSG, oxidized glutathione; GPx, GSH peroxidase; GR, GSSG reductase; GST, GSH transferase; H2-DCFDA, 2¢,7¢-dichlorofluorescin-diacetate; IRP-1, iron regulatory protein1; LDH, lactate dehydrogenase; MFI, mean log fluorescence intensity; MnSOD, manganese superoxide dismutase; MPA, metaphosphoric acid; mt, mitochondrial; NBT, nitroblue tetrazolium; PMRS, plasma membrane oxidoreductase system; PBN, N-tert-butyl-a-phenylnitrone; ROS, reactive oxygen species; SOD, superoxide dismutase Enzymes: catalase (EC 1.11.1.6); GSH peroxidase (EC 1.11.1.9); GSSG reductase (EC 1.8.1.7); GSH transferase (EC 2.5.1.18); Mn superoxide dismutase, CuZn superoxide dismutase, superoxide dismutase (EC 1.15.1.1) (Received 26 April 2004, revised 16 July 2004, accepted 23 July 2004) Keywords: A549 q0 cells; antioxidant defences; 143 q0 cells; reactive oxygen species; rhabdomyosarcoma q0 cells Ó FEBS 2004 complex I and III, or a rise in oxygen tension (state respiration) Defects in respiratory complexes [9] and normal aging [10] also lead to increased mitochondrial ROS production A recent study [11] indicates that mitochondrial ROS homeostasis plays a key role in the life and death of eukaryotic cells, as mitochondria not only respond to ROS but also release ROS in response to a number of pro-apoptotic stimuli However, mitochondria are not the sole source of cellular ROS ROS also form in the cytosol and in peroxisomes as by-products of specific oxidases [7,10] The plasma membrane oxidoreductase system (PMRS) also influences cellular redox state [12,13] Mitochondria are partially autonomous organelles; they possess DNA, which contributes essential proteins to the oxidative phosphorylation system In vitro mammalian cells can be depleted entirely of their mitochondrial DNA, creating so-called q0 cells [14,15] Rho0 cells lack a functional electron transport chain and appear incapable of generating ATP from mitochondria Moreover, it is still a debated question [16] whether or not q0 cells may generate ROS at the mitochondrial level Therefore, q0 cells may require alternative mechanisms for energy supply and for maintenance of an appropriate redox environment [17,18] Analysis of q0 cells has provided insights into oxygen metabolism [13,17,19–21] and the role of mitochondria in redox signalling during apoptosis [22,23] Redox-sensitive signalling and sensitivity to oxidative stress depend on the cell type and its antioxidant systems, due to differential tissue expression of nuclear genes [24] There are no reports that compare antioxidant defences and ROS homeostasis between mitochondrial (mt)DNAdepleted cells with different nuclear backgrounds In this study, soluble and enzymatic antioxidant systems and ROS steady-state level were characterized in three tumour cell lines derived from bone (osteosarcoma, 143B), muscle (rhabdomyosarcoma, RD) and lung (adenocarcinoma, A549) and in the respective q0 cells: 143Bq0 (bone), RDq0 (muscle) and A549q0 (lung) cells This approach was undertaken to investigate the effect of the absence of electron transport chain on cellular redox homeostasis, with the hypothesis that ROS levels could be altered in consequence of the ablation of an efficient respiratory chain We aimed to verify: (a) if q0 status requires antioxidant defence systems as efficient as those of normal q+ cells; (b) if nuclear background influences redox homeostatis in the different cell lines, precursors of cytoplasmic hybrids (cybrids), that are useful tool for studies of mtDNA segregation [25,26] Experimental procedures Materials All reagents and enzymes were from Sigma NaCl/Pi from Oxoid had the following composition: NaCl gỈL)1, KCl 0.2 gỈL)1, Na2HPO4 1.15 gỈL)1 and KH2PO4 0.2 gỈL)1 (pH 7.3) Tissue culture reagents were purchased from Gibco-Invitrogen Co Reverse transcription was performed using the Stratascript enzyme (Stratagene) 2¢,7¢-Dichlorofluorescin-diacetate (H2-DCF-DA) was from Molecular Probes Homeostasis of ROS in q0 cells (Eur J Biochem 271) 3647 Cell lines and culture conditions The q+ wild-type osteosarcoma cells (143B) and the q0 cells derived from 143B were a gift from G Attardi (Division of Biology, California Institute of Technology, Pasadena, CA, USA) [14], RD and RDq0 cells were established by Vergani et al [27], lung carcinoma (A549) and the derived q0 cells were a gift from I J Holt (MRC, Dunn Human Nutrition Unit, Cambridge, UK) [25] The cells were grown in Dulbecco’s modified Eagle’s medium containing 4.5 gỈL)1 glucose, 110 mgỈL)1 pyruvate, supplemented with 10% (v/v) fetal bovine serum, 100 unitsỈmL)1 penicillin, and 0.1 mgỈmL)1 streptomycin, at 37 °C in a humidified atmosphere of 5% CO2 The medium for q0 cells was additionally supplemented with 50 lgỈmL)1 uridine The absence of mtDNA in these three cell lines was reconfirmed at several time points throughout the study by PCR as described previously [14,25,27] Routinely, · 106 q+ or q0 cells were seeded on 100 mm diameter plates and harvested after 42–48 h of culture during the period of exponential growth Subcellular fraction preparation In some experiments regarding aconitase reactivation (see below), 40 · 106 cells suspended in 0.8 mL were treated with digitonin (0.5 mgỈmL)1) in NaCl/Pi for 15 on ice The samples were centrifuged at 17 000 g for 15 at °C, the supernatant (cytosolic fraction) and the pellet (mitochondria-enriched fraction), as well as the whole cells, were recovered, immediately frozen in liquid N2 and stored at )80 °C Aliquots, kept at )80 °C for up to weeks, were thawed immediately before the assay, as reported previously [28] As markers of cytosolic and mitochondria-enriched fractions, lactate dehydrogenase (LDH) [29] and citrate synthase (CS) [30] activities were assayed in total cells and in cytosolic and mitochondria-enriched fractions, respectively In mitochondria-enriched fractions CS activity was twice the value found in the whole cells, whereas cytosolic contamination, checked by measuring LDH, ranged from 10 to 30% In the cytosolic fractions the contamination of mitochondria, checked by measuring CS activity, was about 10% of the value found in whole cells Antioxidant defences Glutathione and oxidized glutathione amounts Cellular glutathione (GSH) and oxidized glutathione (GSSG) levels were measured enzymatically by using a modification of the procedure of Anderson, as described [31,32] The assay is based on the determination of a chromophoric product, 2-nitro-5-thiobenzoic acid, resulting from the reaction of 5,5¢-dithiobis-(2-nitrobenzoic acid) with GSH In this reaction, GSH is oxidized to GSSG, which is then reconverted to GSH in the presence of glutathione reductase and NADPH The rate of 2-nitro-5-thiobenzoic acid formation is measured spectrophotometrically at 412 nm The cells (about 5–6 · 106 cells) were washed once with NaCl/Pi and treated with 6% (v/v) metaphosphoric acid (MPA) (1 mLỈdish)1) at room temperature After 10 the acid extract was collected, centrifuged for at 18 000 g at °C and processed The cellular debris remaining on the plate were solubilized with 0.5 M KOH 3648 L Vergani et al (Eur J Biochem 271) and assayed for their protein content [33] For total glutathione determination, the above acid extract was diluted (1 : 6) in 6% (v/v) MPA; thereafter to 0.1 mL of supernatant, 0.75 mL 0.1 M potassium phosphate, mM EDTA buffer pH 7.4, 0.05 mL 10 mM 5,5¢-dithiobis-2nitrobenzoic acid (prepared in 0.1 M phosphate buffer) and 0.08 mL mM NADPH were added After a equilibration period at 25 °C, the reaction was started by the addition of U glutathione reductase (type III, Sigma, from bakers yeast, diluted in 0.1 M phosphate/EDTA buffer) Product formation was recorded continuously at 412 nm (for at 25 °C) with a Shimadzu UV-160 spectrophotometer The total amount of GSH in the samples was determined from a standard curve obtained by plotting known amounts (from 0.05 to 0.4 lgỈmL)1) of GSH vs the rate of change of absorbance at 412 nm GSH standards were prepared daily in 6% (v/v) MPA and diluted in phosphate/EDTA buffer pH 7.4 For GSSG measurement, soon after preparation the supernatant of acid extract was treated for derivatization with 2-vinylpiridine at room temperature for 60 In a typical experiment, 0.15 mL of supernatant was treated with lL of undiluted 2-vinylpyridine Nine microliters of triethanolamine were also added, the mixture was vigorously mixed, and the pH was checked; it was generally between and After 60 min, 0.1 mL aliquots of the samples were assayed by means of the procedure described above for total GSH measurement The amount of GSSG was quantified from a standard curve obtained by plotting known amounts of GSSG (from 0.05 to 0.20 lgỈmL)1) vs the rate of change of absorbance GSH present in the samples was calculated as the difference between total glutathione and GSSG levels Antioxidant enzyme activities GSH peroxidase (GPx), GSSG reductase (GR), catalase, superoxide dismutase (SOD) and GSH transferase (GST) activities were measured in monolayer cells (about 2–3 · 106 cells), washed three times with NaCl/Pi before treatment directly on the dish with 0.25 M sucrose, 10 mM Tris/HCl pH 7.5, mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM 1,4-dithio-DL-threitol (DTT) and 0.1% (v/v) Nonidet (named solution A), to obtain complete lysis of intracellular organelles Cells were then scraped from the plate and the samples were centrifuged for 30 at 105 000 g Protein content measurements [33] and enzymatic assays were carried out on the clear supernatant fractions Total GPx activity was measured according to the coupled enzyme procedure with glutathione reductase, as described [34], using cumene hydroperoxide as substrate The enzymatic activity was monitored by following the disappearance of NADPH at 340 nm for at 25 °C The incubation medium (final volume mL) had the composition 50 mM KH2PO4 pH 7.0, mM EDTA, mM KCN, mM GSH, 0.1 mM NADPH, U glutathione reductase and % 300 lg protein After a equilibration period at 25 °C, the reaction was started by the addition of 0.1 mM cumene hydroperoxide dissolved in ethanol The specific activity was calculated by using an extinction molar coefficient obtained by a standard curve of NADPH between 0.02 and 0.1 lmolesỈmL)1 and GPx activity was expressed in nmoles NADPH consumedỈmg protein)1Ỉmin)1 Ó FEBS 2004 GR activity was measured according to the method of Carlberg & Mannervik [35], by following the rate of oxidation of NADPH by GSSG at 340 nm for at 25 °C The reaction mixture (final volume mL) contained 0.1 M KH2PO4 pH 7.6, 0.5 mM EDTA, mM GSSG, 0.1 mM NADPH, and % 300 lg protein The specific activity was calculated by using an extinction molar coefficient obtained by a standard curve of NADPH between 0.02 and 0.1 lmolesỈmL)1 and GR activity was expressed in nmoles NADPH consumedỈmg protein)1Ỉmin)1 Total catalase activity was assayed according to the method of Aebi [36] Activity was measured by monitoring, for 30 s at 25 °C, the decomposition of 10 mM H2O2 at 240 nm in a medium (final volume mL) consisting of 50 mM phosphate buffer pH 7.0 and % 100 lg proteins Catalase activity was expressed as unitsỈmg protein)1, assuming that unit of catalase decomposes lmole of H2O2Ỉmin)1 For SOD activity assay a 0.6 mL aliquot of cell lysate was sonicated on ice (2 · 30 s) and centrifuged for 30 at 105 000 g The supernatant was collected and dialysed overnight in cold double-distilled water to remove small interference substances [37] Enzymatic assays were carried out according to the method of Oberlay & Spitz [38], with minor modifications Briefly, in mL 50 mM KH2PO4 pH 7.8 and 0.1 mM EDTA, a superoxide-generating system (0.15 mM xanthine plus 0.02 U xanthine oxidase) was used together with 50 lM nitroblue tetrazolium (NBT) to monitor superoxide formation by following the changes in colorimetric absorbance at 560 nm for at 25 °C The catalytic activities of the samples were evaluated as their ability to inhibit the rate of NBT reduction; increasing amounts of proteins (5–150 lg) were added to each sample until maximum inhibition was obtained SOD activity was expressed as unitsỈmg protein)1, with unit of SOD activity being defined as the amount of proteins causing half-maximal inhibition of the rate of NBT reduction GST activity was assayed in the supernatant of cell lysates, as described [39] Briefly, 150 lg protein were incubated in 50 mM KH2PO4 pH 6.5, mM GSH and 0.25 mM 1-chloro-2,4-dinitrobenzene The reaction was followed for at 37 °C at 340 nm, and GST activity was calculated using an extinction coefficient of 9.6 mM)1Ỉcm)1 [39] Reverse transcription and quantitative PCR RNA (5 lg) was reverse transcribed to cDNA using random hexamer primers and the Stratascript enzyme Quantitative PCR was performed using an MX3000p thermal cycler system and BrilliantÒ SYBER Green QPCR Master Mix (Stratagene) The conditions for the amplification of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and the normalization gene, ribosomal 36B4, were as follows One denaturation step at 90 °C for 10 min, 40 cycles consisting of denaturation at 90 °C for 30 s, annealing at 56 °C for 60 s for CuZnSOD and MnSOD and 60 °C for 36B4, elongation at 72 °C for 60 s At the end of the PCR the samples were subjected to melting curve analysis All reactions were performed in triplicate The primer sequences were CuZnSOD [40], sense 5¢-GCGACGAAG Ĩ FEBS 2004 GCCGTGTGCGTGC-3¢, antisense 5¢-ACTTTCTTCATT TCCACCTTTGCC-3¢; MnSOD [40], sense 5¢-CTTCA GCCTGCACTGAAGTTCAAT-3¢, antisense 5¢-CTGAA GGTAGTAAGCGTGCTCCC-3¢; 36B4, sense 5¢-GTGA TGTGCAGCTGATCAAGACT-3¢, antisense 5¢-GATGA CCAGCCCAAAGGAGA-3¢ Western blot analysis Cells were lysed in the same buffer as used for the enzyme activity assay An equal amount of protein (40 lgỈlane)1) for each sample was separated by SDS/PAGE (12% acrylamide) and transferred to nitrocellulose membrane The membrane was blocked in 5% (w/v) nonfat dry milk in 0.02 M Tris/HCl pH 7.5, 0.137 M NaCl, and 0.1% (v/v) Tween-20 for h at room temperature After overnight incubation at °C in : 1000 of primary antibodies to CuZnSOD (Santa Crutz) or MnSOD (Stressgen Biotechnology), membranes were probed with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) Bound antibody was visualized using an ECL reagent (Amersham Biosciences) Densitometric analysis of Western blot signal was performed using IMAGEMASTER VDS-CL (Amersham Pharmacia Biotech) and IMAGE-MASTER TOTALLAB v1.11 software Homeostasis of ROS in q0 cells (Eur J Biochem 271) 3649 Results The steady-state levels of intracellular ROS depends on the balance between rates of ROS generation and detoxification A crucial role in determining ROS cellular homeostasis is played by the antioxidant defence systems Therefore soluble (GSH and GSSG) and enzymatic defences (GPx, GR, SOD, catalase and GST) were characterized on three human tumour cell lines, with (q+) and without (q0) mtDNA GSH concentration was significantly decreased in all three mtDNA depleted cell lines compared to parental lines with mtDNA; the decrease in GSH content was most pronounced in bone 143B q0 cells (Fig 1) GSSG was also lower in q0 cells compared with q+, but only statistically significant in bone-derived cells (Fig 1) The percentage of ROS measurement Aconitase determination Aconitase activity was measured as described previously [41] on · 106 cells or on the subcellular fractions obtained as reported above The samples were dissolved in 0.1% (v/v) Triton X-100 and incubated for 15 at 30 °C in 50 mM Tris/HCl pH 7.4, 0.6 mM MgCl2, 0.4 mM NADP, mM Na citrate To start the assay, U isocitrate dehydrogenase were added and activity was measured by monitoring absorbance at 340 nm for 15 Reactivation of aconitase was obtained by adding 50 lM DTT, 20 lM Na2S and 20 lM Fe(NH4)2(SO4)2 directly into the cuvette, just before spectrophotometric determination [41] DCF fluorescence Direct detection of intracellular steadystate levels of ROS was carried out on living cells using 2¢,7¢dichlorofluorescin-diacetate (H2-DCF-DA) [42–44] The probe is de-acetylated inside the cell The subsequent oxidation by intracellular oxidants yields a fluorescent product, 2¢,7¢-dichlorofluorescin (DCF) Cells were collected by trypsinization and centrifuged for at 800 g The pellet was incubated in tissue-culture medium with lM H2-DCF-DA for 30 at 37 °C Cells were washed and then suspended (1 · 106 per mL) in medium (standard growth conditions) or in NaCl/Pi for 90 (stress conditions) A FACSCalibur analyser (Becton-Dickinson Immunocytometry Systems) equipped with a 488 Argon laser was used for measurements of intracellular fluorescence Dead cells were excluded by electronically gating data on the basis of forward- vs side-scatter profiles; a minimum of · 104 cells of interest were analysed further Logarithmic detectors were used for the FL-1 fluorescence channel necessary for DCF detection Mean log fluorescence intensity (MFI) values were obtained by the CELLQUEST software program (Becton-Dickinson) Fig GSH and GSSG concentrations and ratio of GSH2 : GSSG in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q+ value at: *P < 0.05; **P < 0.01 3650 L Vergani et al (Eur J Biochem 271) mitochondrial GSH in respect to total GSH was similar in all tested q+ and q0 cell lines, ranging from 2.7 to 5% (data not shown) To assess the cellular redox state we measured the GSH2 : GSSG ratio which is considered a good index of this parameter [45] MtDNA loss was associated with an alteration in this ratio with q0 cells having a more oxidized redox state than q+ cells However the change was statistically significant only in bone-derived q0 cells Moreover, the different values found in bone, muscle and lung q0 cells were all significantly different (P < 0.05) from each other; in fact the GSH2 : GSSG ratio of bone 143Bq0 cells is about one-half of that in muscle RDq0 cells and even three to four times lower than that measured in lung A549q0 cells GPx and GR are crucial antioxidant defences as GPx transforms H2O2 to H2O by coupling the oxidation of GSH to GSSG and GR mediates the reduction of GSSG to GSH In the three cell lines tested, mtDNA loss was associated with a four-fold increase in GPx activity and a significant decrease in GR activity (Fig 2) Moreover Fig shows that the absolute values of GPx and GR activity were considerably higher in lung q0 cells than in other q0 cells (Fig 2) Catalase activity was assessed in q+ and q0 cells; our findings show that such activity was not affected by mtDNA depletion (data not shown) Activity, gene expression and protein content of SOD were studied Total SOD activity was decreased in bone and muscle q0 cells compared with their parental q+ lines Fig GPx and GR activities in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q+ value at: **P < 0.01; ***P < 0.001 Ó FEBS 2004 Fig Total SOD activity in q+ and q° cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q+ value at: ***P < 0.001 (Fig 3), whereas there were no significant differences in the activity and expression levels in lung q+ and q0 cells (Figs 3–5) Quantitative PCR (Fig 4) and Western blot (Fig 5) analysis were carried out to evaluate the relative contribution of MnSOD and CuZnSOD Both analyses confirmed that bone q0 cells had significantly lower expression of CuZnSOD than the other cells In musclederived cell lines mtDNA ablation reduced the expression and protein amount of mitochondrial MnSOD but not of cytosolic CuZnSOD (Figs and 5) Densitometric analysis of Western blot was in line with the results of quantitative PCR (data not shown) Glutathione S-transferase (GST) enzymes metabolize xenobiotics as well as aldehydes, endogenously produced during lipid peroxidation, by conjugation with GSH Moreover, some GSTs also show glutathione-peroxidaselike activity [1] GST activity was decreased to a similar extent in bone- and muscle-derived q0 cells, compared with the parental q+ cells, but the absolute value was significantly higher in bone than in muscle q0 cells No differences were evident in lung q+ and q0 cell lines (Fig 6) To check the ability of the antioxidant defences to balance ROS generation, indirect and direct measurements of intracellular steady state levels of ROS were performed Indirect measurements were carried out by assessing the aconitase activity Aconitase is a four iron–sulfur cluster (Fe–S)containing hydratase, present in various subcellular compartments (i.e mitochondria and cytosol) which is inactivated by À [41] In the cytosol, loss of aconitase activity results in the conversion of this enzyme to the iron regulatory protein-1 (IRP-1), that serves to regulate iron homeostasis [46], and mitochondrial aconitase inactivation serves as a protective response to oxidative stress [46] Aconitase activity was measured in q+ and q0 cell lines under basal culture conditions and after 18 h of treatment with the ROS spin-trapping N-tert-butyl-a-phenylnitrone (PBN) [47,48] Figure shows a trend of increasing aconitase activity in almost all PBN-treated cell lines The increase was most marked in bone q+ and q0 cells (more Ó FEBS 2004 Homeostasis of ROS in q0 cells (Eur J Biochem 271) 3651 Fig Western blotting analysis of CuZnSOD and MnSOD in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Total cell extract was resolved by SDS/PAGE and blotted onto nitrocellulose The membrane was cut in strips, corresponding to the different molecular masses of MnSOD, CuZnSOD and actin, the last acting as an internal standard, and incubated with the corresponding antibody Forty micrograms of cell protein extract was loaded in each lane The blots depicted are representative of three separate experiments Fig Quantitative real-time PCR of CuZnSOD and MnSOD in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) mRNA values of CuZnSOD and MnSOD are normalized for ribosomal 36B4 gene and are expressed as means ± SD of three assays in triplicate in arbitrary units (A.U.) Significant differences from respective q+ value at: *P < 0.05 than five-fold) and in muscle q0 cells, suggesting that the À level was higher in these cells than in lung q0 cells Both mitochondrial [28,46] and cytosolic IRP-1/aconitase activities [46] are reactivated in the presence of reducing agents and free Fe2+ carrier–donor [41] Therefore, in an attempt to localize À production, we assessed aconitase reactivation in these subcellular fractions Reactivated aconitase showed a dramatic increase in cytosolic fractions of bone q0 cells (Fig 8), whereas in mitochondria-enriched fractions there were no significant differences Lastly, by means of the DCF technique coupled to flow cytometric analysis, intracellular fluorescence was measured as an index of steady-state levels of ROS under basal and stress conditions (Fig 9, Table 1) In the presence of glucose and 10% serum (standard growth conditions), the fluorescence measured in q0 cells was lower than that in the parental cell lines containing mtDNA The decrease was substantial in lung (90%) and muscle (40%) cells but was less evident in bone (less than one-third) (Table 1) When the cells were incubated in NaCl/Pi for 90 min, the intracellular fluorescence signal dramatically increased in all cases (Fig 9, Table 1) The increases, in comparison to the signals observed in standard growth conditions, were Fig GST activity in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q+ value at: **P < 0.01 consistently greater in q0 than in q+ cells, yet the extent of the increase varied considerably between the three q0 lines In bone and lung q0 cells the increases were 17- and 39-fold, respectively However only in bone q0 cells was DCF oxidation significantly higher compared to the value of the respective q+ cell line (Table 1) Discussion Our analysis of three pairs of q+ and q0 cells, derived from bone, muscle and lung, indicates that these cells differ significantly both in their antioxidant defences and intracellular ROS homeostasis The antioxidant system is 3652 L Vergani et al (Eur J Biochem 271) Ó FEBS 2004 Fig Aconitase activity in whole cells in absence (–) and presence (+) of PBN Rho+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) were cultured in the absence (–) or the presence (+) of 500 lM PBN for 18 h Aconitase activity were assayed spectrophotometrically in cell lysate Values are expressed as means ± SD of at least three assays in duplicate as nmolesỈmin)1Ỉmg)1 protein –PBN value significantly different from +PBN value at: *P < 0.05; **P < 0.01; ***P < 0.001 profoundly affected by mtDNA depletion in a tissue specific-manner, probably as a response to a decreased need of efficient antioxidant machinery + Antioxidant defences of parental q cell lines The parental (q+) A549 cells, derived from type II human alveolar epithelial cells [49], are provided with the highest GSH content and GSH2 : GSSG ratio (Fig 1), and the highest GPx, GR (Fig 2) and SOD (Fig 3) activities in comparison with bone and muscle derived q+ cells This very efficient ROS defence system may be related to the high oxygen tension normally present in the lung and explains the great resistance of these cells to apoptosis, after exposure to high oxygen concentrations [50] By contrast, bone (143B)- and muscle derived (RD)- cells are similar in their low content of GSH (only one-half of that present in A549) and poor GPx activity (Figs and 2); however, RD cells differ significantly in GR activity and in particular in activity, gene expression and protein content of SOD (Figs 3–5) Antioxidant defences of q0 cell lines GSH-GSSG and GR We measured GSH and GSSG in exponentially growing cells, as GSH content changes in the growth and lag phases [51] In all q0 cells studied, GSH was significantly lower than in the respective parental cells, with the lowest GSH level in bone-derived q0 cells, and significant differences in the GSH2 : GSSG ratios among the different q0 cells (Fig 1) The intracellular content of GSH is the result of balance between its synthesis and consumption GSH synthesis is a two-step ATP-requiring process catalysed by cytosolic c-glutamylcysteine synthetase (c-GCS) and GSH synthetase and is regulated (feedback-inhibited) by GSH itself [52] We neither directly measured these Fig Aconitase reactivation Aconitase activity was assayed in mitochondrial and cytosolic fractions of q+ and q0 from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Reactivation was achieved in presence of reducing agents (DTT) and Fe2+ carrier–donor [Fe(NH4)2(SO4)2], as described in Experimental procedures, and is expressed as percentage of basal value Basal values (nmolesỈmin)1Ỉmg protein)1) of mitochondrial aconitase activity were: in bone q+ ¼ 3.26 ± 1.87 (4); bone q0 ¼ 2.36 ± 0.93 (4); muscle q+ ¼ 8.77 ± 0.57 (3); muscle q0 ¼ 2.08 ± 0.19 (3); lung q+ ¼ 8.46 ± 4.12 (3); lung q0 ¼ 4.88 ± 0.59 (3) Basal cytosolic aconitase in bone q+ ¼ 1.64 ± 0.57 (4); bone q0 ¼ 2.81 ± 1.12 (4); muscle q+ ¼ 0.76 ± 0.29 (3); muscle q0 ¼ 1.26 ± 0.53 (3); lung q+ ¼ 4.79 ± 0.6 (3); lung q0 ¼ 4.59 ± 2.27 (3) Significant differences from respective q+ value at: *P < 0.05, **P < 0.01 activities in our q0 cells nor did we find reports on this topic in the literature, but we did find a very low amount of ATP (data not shown) in all of the q0 cells compared with the respective parental q+ cells The smaller GSH pool in q0 cells (reduced GSH and GSSG) suggests that it could be due to reduced synthesis rather than to enhanced utilization in cells with low amounts of ATP In fact if the lower level of GSH in q0 cells was due to its extensive consumption in the GPx pathway or to a direct interaction with ROS, we should find increased GSSG In our experimental conditions we found that GSSG levels in all q0 cell lines were not increased, but rather decreased, although GR activity was significantly decreased in all q0 cells (Fig 2) However, it cannot be excluded that GSSG is actively secreted from the cells subjected to an oxidative stress [52] in an attempt to maintain cellular redox environment [45] Therefore our data could indicate that mtDNA-depleted cells need less Homeostasis of ROS in q0 cells (Eur J Biochem 271) 3653 Ó FEBS 2004 ρ+ 103 104 101 102 FL1-H 103 104 101 102 103 104 103 104 101 102 FL1-H 103 104 101 102 FL1-H 103 104 60 40 Counts 20 80 100 60 40 20 0 100 102 FL1-H 100 Counts 60 40 20 Counts Lung 101 80 100 100 80 100 100 60 80 100 20 0 Counts 102 FL1-H Muscle 101 30 60 90 120 150 180 100 40 Counts 60 20 Bone 40 Counts 80 100 ρ0 FL1-H Blank Standard growth condition Stressed condition 100 Fig DCF oxidation in cells with and without glucose Rho+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle), and lung carcinoma (lung) were collected and loaded with H2-DCF-DA Fluorimetric signals of oxidized DCF (excitation, 488 nm; emission, 530 nm) were recorded by cytofluorimeter from cells in presence of glucose (dotted line): standard growth conditions or in absence of glucose (bold line): stressed conditions Blank signal, obtained from cells without H2-DCF-DA, was deducted to the reported MFI values The panels are representative of the separate experiments summarized in Table Table Levels of DCF oxidation in q+ and q0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) MFI of the DCF signal was measured by fluorescence activated cell sorting as arbitrary units in cells in presence of glucose (standard growth conditions) and in absence of glucose (stress conditions) Values are expressed as mean ± SD as arbitrary units of fluorescence Numbers in parentheses are the numbers of experiments Significant differences from respective q+ value: *P < 0.05; ***P < 0.001 Conditions Standard growth Bone Muscle Lung a q+ q0 q+ q0 q+ q0 Stressa 186 142 208 143 235 25 1275 2500 1055 996 1693 976 ± ± ± ± ± ± 33 (4) 75 (6) (3) (3)*** 13 (3) (3)*** ± ± ± ± ± ± 92 (3) 217 (3)* 315 (3) 210 (3) 245 (3) 319 (3) P < 0.001 vs respective values in standard growth conditions anti-ROS buffer in the form of GSH for loss of ROS mitochondrial fluctuation and of ROS spike, occurring when the respiratory chain is active SOD, GST, GPx and catalase With the exception of catalase and GPx activity, depletion of mtDNA diminished SOD and GST activities in boneand muscle-derived q0 cells but not in lung-derived q0 cells (Figs 3–6), where SOD (Figs 3–5) and GST (Fig 6) were unaffected after ablation of the respiratory chain In bone and muscle q0 cells SOD activity decreased (Fig 3) as compared with the respective parental q+ cells Expression level analysis revealed that in bone q0 cells CuZnSOD mRNA (Fig 4) and protein content were decreased (Fig 5), whereas in muscle q0 cells MnSOD decreased in mRNA and protein amount compared with parental cells (Figs and 5) The decrease of SOD and GST antioxidant enzymes in bone and muscle but not in lung q0 cells might be ascribed to different expression–regulation of nuclear genes as a response to cell type differential redox-sensitive signalling [53] Catalase activity is unaffected by mtDNA depletion (data not shown) and, interestingly, the activity of GPx was found to be considerably increased in all q0 cells relative to the parental cells (Fig 2) GPx, together with catalase and thioredoxin peroxidase, restricts H2O2 accumulation and the consequent production of highly reactive Ó FEBS 2004 3654 L Vergani et al (Eur J Biochem 271) hydroxyl radicals, for which no physiological defence system exists [1] In the last few years, the view of hydrogen peroxide as a merely toxic by-product of cellular metabolism has changed, and it is now recognized as playing an important role in intracellular signalling [3–5] Fine regulation of redox balance may therefore be a critical function of peroxidases, catalase and of GPx, in particular [54] GPx regulates the intracellular hydroperoxides and lipid hydroperoxides used as signal transducers of many transcription factors including nuclear factor-jB [55], AP-1 [56] and MAP kinases [57] Because catalase is unchanged, the increased GPx activity of q0 cells may be an essential cellular adaptation that enables gene expression to function normally in the absence of mtDNA These findings are in line with results found in hepatoma-derived Hep1q0 cells [16] ROS When DCF signal was assessed as a direct index of ROS, all of the q0 cells had a reduced intracellular fluorescence compared to q+ cells Bone-derived q0 cells had the highest level of intracellular ROS compared to muscle and lung q0 cells both in standard growth conditions and in stressed conditions (Fig 9, Table 1) If the current idea, that the DCF technique mainly determines cellular peroxides [42– 44,58], is accepted it can be hypothesized that q0 cells accumulate a lower DCF fluorescence signal due to their high GPx activity (Fig 2) in a tissue-specific manner In fact, lung q0 cells have the lowest DCF oxidation (Fig 9, Table 1) and the highest GPx activity (Fig 2), whereas bone- and muscle-derived q0 cells have rather similar GPx activities and similar capacities to eliminate intracellular oxidants under standard growth conditions Yet, in the absence of glucose (stress conditions), intracellular levels of ROS in bone-derived q0 cells are 2.5 times those of muscle q0 cells (Fig 9, Table 1) This may be due to the fact that among q0 cells, bone q0 cells had the less efficient antioxidant machinery with the lowest GSH level (Fig 1) Interestingly, bone-derived q0 cells also featured the highest glucose consumption rate and glucose-6-phosphate dehydrogenase activity among the six lines analysed (L Vergani, unpublished data) Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme in the pentose phosphate pathway and a major source of cytosolic NADPH and ribose phosphate [59] When glucose is scarce, NADPH synthesis decreases This lead to a decrease in GSH levels as NADPH is required for GSH regeneration via GR Therefore, our data suggest that increased generation of intracellular ROS in bone q0 cells, relative to muscle q0, is due to increased production of oxidants The high production of ROS in bone-derived q0 cells is further confirmed by indirect measurement of ROS obtained by comparing aconitase activity in standard conditions and after 18 h of incubation with PBN (Fig 7) In biological systems PBN [60,61], or N-t-butyl hydroxylamine, a breakdown product of PBN [47,48], efficiently trap free radicals, such as superoxide anion (2 À ) that in turn inactives aconitase [41] The observed PBN-induced increase in aconitase activity in bone q+ and q0 cells and in muscle q0 cells (Fig 7) strongly supports a high presence of À in these cells also in standard growth conditions These data are well related to the lowest GSH2 : GSSG ratio and the most oxidized redox state (Fig 1) A PBN effect on antioxidant enzyme activities may be excluded on the basis of a recent report showing that PBN protects U937 cells against ionizing radiation-induced oxidative damage by altering cellular redox state but not affecting antioxidant enzymes [61] New and original evidence emerges from the experiments of reactivation of aconitase activity by reducing agents and Fe(NH4)2(SO4)2, as a Fe2+ carrier–donor [41] Figure shows a dramatic increase in cytosolic IRP-1/aconitase activity in bone q0 cells, but not in mitochondria-enriched fractions This finding suggests that in bone q0 cells intracellular oxidants derive chiefly from nonmitochondrial compartments and are therefore not related to a vestige of the respiratory electron transport chain Possible sources of nonmitochondrial oxidants include NADPH oxidases [12], and lipoxygenases, whose action plays a role in signal pathways of growth factor-stimulated bone cell mitogenesis [62], and microsomal redox systems [63] NADPH oxidases are up-regulated in lymphoblastoid q0 cells, as a compensatory phenomenon in maintaining cell viability [18] Our results confirm PMRS as a possible source of ROS in bone cells, as the NADPH oxidase inhibitor diphenyleniodonium chloride reduces fluorescence accumulation into bone q+ and q0 cells to 65–70% (data not shown) Another possible explanation for the increased generation of intracellular oxidants in bone-derived q0 cells is the high O2 tension to which cultured cells are exposed compared to the low O2 tension of osteoblasts The bulk of intracellular oxidants in bone-derived q0 cells is in extra-mitochondrial compartments, corroborating an earlier report which showed q0 cells to be sensitive to the ablation of cytosolic SOD [64] Moreover the presence of extramitochondrial ROS in q0 cells could explain the similar levels of oxidative DNA damage observed in Hela q0 and the parental q+ cells [65] In conclusion, our study demonstrates that loss of functional mitochondria, the major cellular site for ROS formation, reduces enzymatic and soluble intracellular antioxidant defences but not ROS flux in the studied q0 cells, and that there are cell line-to-cell line variations in intracellular antioxidant defences and ROS homeostasis In fact among the studied cells, those originating from bone are particularly vulnerable to free radical-induced stress after mtDNA ablation These differences could reflect tissuespecific aspects of intracellular oxidant metabolism, although it is inevitable that some specific features of ROS homeostasis in terminally differentiated tissues such as bone, lung and muscle will have been lost during the transformation process that led to tumour formation The pronounced difference in intracellular homeostasis between lung A549 and bone 143B q0 cells may also be germane to mtDNA segregation bias, as selection of mutant and wildtype mtDNA is 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(Figs 3–5) Antioxidant defences of q0 cell lines GSH-GSSG and GR We measured GSH and GSSG in exponentially growing cells, as GSH content changes in the growth and lag phases [51] In all q0 cells studied,... cells, and that there are cell line-to -cell line variations in intracellular antioxidant defences and ROS homeostasis In fact among the studied cells, those originating from bone are particularly... Determination of intracellular reactive oxygen species as function of cell density Methods Enzymol 352, 91–100 44 Zuo, L & Clanton, T.L (2002) Detection of reactive oxygen and nitrogen species in