A. Martinuzzi, ‘E. Medea’ Scientific Institute, Conegliano Research Centre, via Costa Alta 37, 31015 Conegliano (TV), Italy E-mail: Andrea.email@example.com
Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain127, 2183–2192]. The aim of this study was to characterize the antioxidant defences of these LHON-affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose-rich medium and galactose-rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.
Leber's hereditary optic neuropathy (LHON), the first disease to be linked with a maternally inherited mtDNA point mutation , is a genetic form of retinal ganglion cell degeneration leading to loss of central vision and optic nerve atrophy occurring predominantly in young males . Three pathogenic mutations at nucleotides 11778, 3460 and 14484, affecting, respectively, ND4, ND1 and ND6 subunit genes of complex I of the respiratory chain, are most often associated with the disease, even though other rare pathogenic mutations have been reported . Complex I dysfunction is postulated to underlie LHON pathogenesis. However, the details of complex I dysfunction in LHON and its consequences on cellular function, with particular reference to the specific retinal ganglion cell degeneration, are still poorly understood.
The biochemical phenotype of complex I dysfunction in LHON has been investigated in various patient-derived tissues (lymphocytes, platelets, muscle) and cell lines (fibroblasts and lymphoblasts) (reviewed in ). Spectrophotometrically assessed complex I specific activity was essentially slightly affected by the 11778/ND4 mutation and not at all by the 14484/ND6 mutation, but the 3460/ND1 mutation consistently lowered the enzyme activity [4–6].
A useful experimental approach for the study of the cellular phenotype associated with the LHON mutations is provided by the transmitochondrial cytoplasmic hybrid (cybrid) cellular model [6–9]. Cybrids are obtained by fusing a rho° cell line (host) completely devoid of mtDNA with cytoplasts produced by enucleation of cells (donor) derived from patients or controls . In this way, the mtDNA of a donor cell can be studied in the context of a ‘neutral’ nuclear background.
The current knowledge suggests that the 3460/ND1 and 11778/ND4 mutations consistently decrease complex I-driven respiration, whereas the 14484/ND6 mutation induces a milder defect . On the other hand, complex I-dependent ATP synthesis is severely affected in cybrids with all three common LHON mutations (reviewed in ). However, the limited clinical expression of LHON suggests that the total ATP cellular content is compensated in most tissues. Concurrently, there is a potential for stably increased production of reactive oxygen species (ROS) [2,3,12]. Indeed an excess of ROS production, in particular mitochondrial superoxide anion, has been observed in neuronal (NT2) cybrid cells carrying the 11778/ND4 and 3460/ND1 LHON mutations, after retinoic acid-induced differentiation  and, more recently, in osteosarcoma-derived cybrid cell lines carrying the three pathogenic mutations 11778/ND4, 3460/ND1 and 14484/ND6 .
Under such conditions, oxidative stress may become the prevalent pathological consequence of complex I dysfunction and trigger apoptotic cell death . In accordance with this view, two recent studies using different models showed the convergent result that LHON pathogenic mutations predispose cells to apoptosis [16,17].
As oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in LHON pathogenesis, the aim of this study was to investigate the level and efficiency of antioxidant defences in cells carrying the most common LHON mutations. Therefore, mitochondria carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations were used to generate osteosarcoma-derived cybrids. In these cells, in which we observed different extents of reduced oxygen consumption, we measured the basal content of glutathione (GSH), oxidized glutathione (GSSG) and the activities of the antioxidant enzymes glutathione peroxidase (GPx; EC 18.104.22.168), glutathione reductase (GR; EC 22.214.171.124), superoxide dismutase (SOD; EC 126.96.36.199) and catalase (EC 188.8.131.52). For comparison, we also carried out all determinations on cybrids repopulated with control mitochondria. Furthermore, we measured the same parameters in the same cybrids subjected to glucose deprivation and galactose replacement in the culture medium, an experimental condition shown to accelerate apoptotic death in cells bearing LHON mutations . As under these conditions the cells are forced to rely on mitochondrial respiratory chain for their ATP production, glucose replacement with galactose represents an ideal system for studying a response to metabolic/oxidative stress in cells showing impaired mitochondrial function.
Oxygen consumption in control and LHON cybrids
The results reported in Fig. 1 allow a comparison of oxygen consumption between multiple controls and LHON cybrids. Grouping all control cybrids, and grouping LHON cybrids by pathogenic mutation, it is evident that mitochondrial respiration is significantly decreased in LHON cybrids carrying the most severe 11778/ND4 and 3460/ND1 point mutation, the mean reduction in respiration compared with the controls being 29.3% (P < 0.001) and 33.5% (P < 0.001), respectively. In contrast, in 14484/ND6 affected cybrids the reduction was only 8.9%[P = nonsignificant (NS)], confirming the milder phenotype of the 14484/ND6 mutation. As no detectable differences were observed among the data obtained from different clones of the same cell line, or among data obtained in single controls and in single cell lines affected by the same mtDNA mutation, we decided to carry out the following experiments in a single clone representative of each independent line (HPC7, control; HFF3, 11778/ND4; HMM12, 3460/ND1; HL180, 14484/ND6).
Antioxidant defences in cybrids incubated in glucose medium
The pattern of antioxidant defences was evaluated in cybrids maintained in basal culture conditions, i.e. in the presence of high glucose concentration (25 mm; glu-) in the medium, which was Dulbecco's modified Eagle's medium (DMEM). The data reported in Table 1 clearly indicate that both GSH and GSSG concentrations were similar in all cybrids tested. GSH concentration in cells bearing 3460/ND1 and 11778/ND4 mutations tended to be lower than in other cybrids, but the observed differences were not statistically significant. As expected, in basal conditions, all cells maintained a very high ratio between reduced and oxidized glutathione, the percentage of GSSG with respect to total glutathione (GSH + GSSG) being about 0.4 to 0.5% in all cell lines.
Table 1. GSH and GSSG content (nmol per mg protein) in control and LHON-affected cybrid cells cultured in glu-DMEM. The results are means ± SD from four independent experiments carried out in duplicate on two dishes for each experiment.
% GSSG/ (GSH + GSSG)
38.90 ± 4.10
0.16 ± 0.04
40.88 ± 6.01
0.15 ± 0.04
34.16 ± 4.16
0.13 ± 0.01
35.29 ± 6.16
0.18 ± 0.12
In Fig. 2 the activities of the glutathione-related enzymes, GPx and GR, are reported. Total GPx activity measured in cells with a 3460/ND1 mutation was less than half of the activity present in controls. In contrast, there were no significant differences from the controls in the enzymatic activities of cells with 14484/ND6 or 11778/ND4 mutations. GR activity in cells carrying the 3460/ND1 or the 11778/ND4 mutation was significantly lower than in controls or cybrids carrying the 14484/ND6 mutation. Also in this case, the activity present in cells bearing the mild 14484/ND6 mutation was not different from that in control cybrids.
When we assessed the activity of catalase, we did not find any significant difference among the various cybrid lines (data not shown).
To complete the analysis of antioxidant enzyme profile in cybrids maintained in glu-DMEM, cytosolic CuZnSOD and mitochondrial MnSOD were measured. CuZnSOD and MnSOD protein in control and LHON-affected cybrids was quantified by Western blot using a specific antiserum (Fig. 3). Densitometric analysis of the blots (Fig. 4A) shows a trend of increase in CuZnSOD and MnSOD in LHON-affected cybrids with respect to controls, the difference being significant (P < 0.01) for CuZnSOD in cells carrying the 11778/ND4 mutation. However, the high expression of SOD proteins in cybrids with LHON-associated mutations did not correspond to higher enzymatic activities. The results reported in Fig. 4B in fact indicate that CuZnSOD activity (clear columns) tended to be lower in all cybrid lines compared with controls, whereas MnSOD activity (dotted columns) was significantly (P < 0.05) lower in cells bearing the 14484/ND6 mutation. When CuZnSOD and MnSOD activities were normalized to the respective protein amounts, assessed as densitometric units, the activities of the enzymes were always lower (P < 0.05) in mutated cybrids than controls (Fig. 4C).
Antioxidant defences in cybrids incubated in galactose medium
GSSG concentrations were measured during a 24-h time course experiment in cells cultured in gal-DMEM (Fig. 5) and, for comparison, in glu-DMEM. In the latter condition, GSSG did not change over time (data not shown). In contrast, a marked time-dependent increase in GSSG concentration was observed in cybrids cultured in galactose. Controls and 14484/ND6 mutated cells showed similar behavior over time; after 10 to 16 h of incubation in gal-DMEM, GSSG concentrations had increased significantly (P < 0.001) compared with the respective values observed in glu-DMEM (data not shown). In contrast, in 3460/ND1 mutated cells, GSSG had increased significantly (P < 0.05) after 6 h of treatment, and after 24 h the GSSG concentration was about 30-fold higher than that measured in glucose medium. Moreover, starting at 6 h of incubation in galactose, these cells had significantly (P < 0.001) higher GSSG concentrations than those measured at the same times in controls and 14484/ND6 mutated cybrids. The increase in GSSG concentration was even more marked in 11778/ND4 affected cells; with respect to the concentrations found in glucose-treated cells, the increase in GSSG began to be significant (P < 0.01) after 2 h of treatment and peaked after 16 h, reaching a 45-fold increase. Between 2 and 16 h of the galactose challenge, GSSG concentrations in these cybrids were significantly (P < 0.001) higher than those measured at the same times in all other cybrids. Compared with other mutant cybrids, the GSSG concentration in cells with the 11778/ND4 mutation tended to decrease after 16 h of treatment, possibly indicating a severe cellular defect.
Cellular GSH did not decrease as a consequence of glucose deprivation, but rather increased in some cybrid lines (Fig. 5). In both controls and cells with the 14484/ND6 mutation, GSH concentrations measured after treatment with gal-DMEM were not significantly different from those obtained in glu-DMEM (data not shown), and no significant differences were observed at any time in GSH concentrations between controls and 14484/ND6 affected cybrids in gal-DMEM. In contrast, GSH markedly increased in cells carrying the 3460/ND1 and 11778/ND4 mutations, which once again showed similar behavior. A 12-h incubation in galactose caused a significant (P < 0.01 to 0.001) increase in GSH concentration compared with the values observed in glu-DMEM (data not shown). The GSH concentration in both cell lines had doubled after 16 to 24 h in gal-DMEM and was significantly (P < 0.01 to 0.001) higher than in controls and 14484/ND6 affected cells starting at 10 h of the galactose challenge. However, in spite of this marked increase in GSH concentration, the percentage of GSSG with respect to total glutathione (GSSG + GSH) in cybrids carrying the 3460/ND1 and 11778/ND4 mutations was significantly higher than that measured in controls and cells with the 14484/ND6 mutation (Table 2), indicating a large imbalance in glutathione homeostasis and conditions of extreme oxidative stress in these cells.
Table 2. Percentage of GSSG vs. (GSH + GSSG) in control and LHON-affected cybrid cells cultured in gal-DMEM for 6, 12 or 24 h. The results, obtained from GSSG and GSH values reported in Fig. 5 are means ± SD from four independent experiments carried out in duplicate on two dishes for each experiment.
% GSSG/(GSH + GSSG)
Significant difference from respective control value: P < 0.05.
When we measured the activities of the antioxidant enzymes in cybrids maintained for 24 h in galactose medium, we found that GPx and catalase activities were not different from those in basal conditions (data not shown). In contrast, in some cybrid lines, GR and SOD activities were significantly affected by glucose deprivation. The results reported in Fig. 6 indicate that the cells carrying the 3460/ND1 or 11778/ND4 mutation had significantly lower GR activity than controls and cybrids with the 14484/ND6 mutation. Comparison of these results with those reported in Fig. 2 shows that glucose deprivation caused a further marked decrease in the already low GR activity present in the 11778/ND4 mutated cells, whereas the trend of GR activity in the other cell lines was similar to that observed when the cells were incubated in normal glucose medium.
Incubation in galactose caused a dramatic decrease in SOD activity, particularly mitochondrial MnSOD, in all LHON cybrids (Fig. 7). This decrease ranged from 37% in the cells with the 14484/ND6 mutation to 68% in those with the 11778/ND4 mutation, compared with the controls. Comparison of the results reported in Fig. 7 with the data in Fig. 4B clearly indicates that MnSOD activity in control cells was not affected by the incubation in gal-DMEM.
CuZn-SOD activity was less affected by glucose deprivation. In gal-DMEM, only the 3460/ND1 mutated cells had CuZn-SOD activity significantly different from that in controls, whereas in all other lines it was similar to that observed in glu-DMEM (for comparison, see Fig. 4B).
A complex I-driven chronic increase in oxidative stress has been suggested to be a relevant contributory factor to retinal ganglion cell death and optic atrophy in LHON [2,3,12]. The present results indicate that osteosarcoma-derived cybrids carrying the three most common LHON pathogenic mutations in complex I subunit genes show a partial respiratory defect, assessed as a decrease in oxygen consumption, closely related to the severity of the clinical spectrum of the disease [2,3]. In fact, a 29 to 34% decrease in cell respiration is observed in cells bearing the 11778/ND4 and 3460/ND1 mutations, whereas a lower (≈ 9%) decrease in oxygen consumption is present in cybrids with the 14484/ND6 point mutation, compatible with the milder clinical phenotype . In the same cybrids, a significant increase in ROS production has recently been reported ; in particular, the highest ROS production was measured in cybrids bearing the 3460/ND1 mutation, followed by 11778/ND4 and 14484/ND6 mutations. In this study, we also show, for the first time, that in these LHON-affected cells there is low efficiency of the antioxidant machinery, the 11778/ND4 and 3460/ND1 mutations expressing clearly the most severe phenotype. The greatest vulnerability of these cells to metabolic/oxidative stress is magnified by glucose deprivation and galactose replacement.
Antioxidant defences in cybrids cultured in glu-DMEM
We first assessed the antioxidant defences of LHON cybrids in glucose-supplemented medium (glu-DMEM), a condition in which cells derive their energy mainly from anaerobic glycolysis  and ATP production is ensured even in the presence of mitochondrial dysfunction [19,20]. Under these culture conditions, LHON cybrids grew normally, as previously reported [7,17]. However, some alterations in their antioxidant machinery emerged, more clearly in cybrids carrying the 3460/ND1 and 11778/ND4 mutations in which GPx and GR activities were significantly reduced. As GPx and GR are proteins encoded by the nuclear genome of the parental 143B.TK– cells, which is constant among the cybrid cell lines compared in this study, the low GPx and GR activities in 3460/ND1 and 11778/ND4 mutated cybrids may be ascribed to post-translational events. It is well known that GR [21,22] and GPx  activities are significantly decreased in the presence of ROS or in a condition of drug-induced ROS generation. As increased generation of ROS has been observed in the glu-DMEM cultured cybrids, particularly 3460/ND1 and 11778/ND4 mutated cells , we can hypothesize that the decrease in GPx and GR activities displayed by these mutants may reflect the higher ROS concentrations. On the other hand, significant increases in ROS production have also been observed in NT2 neuronal-like cybrids carrying the 11778/ND4 and 3460/ND1 mutations  and in human–ape xenomitochondrial cybrids partially deficient in complex I . A ‘chronic’ oxidative insult may also explain the apparent contradiction of SOD results in LHON cybrids. As shown by Western blot analysis, the expression of CuZnSOD and MnSOD proteins seems to be slightly increased in LHON cybrids compared with controls, whereas the enzyme activities are lower. Both MnSOD and CuZnSOD proteins are possibly upregulated as a compensatory mechanism, but may be partially inactivated by oxidative damage. Complex I impairment in a variety of human diseases has been previously reported in association with MnSOD induction, although not all patients defective in complex I displayed an increase in MnSOD activity . Furthermore, not all tissues have the same ability to upregulate MnSOD, as suggested by the different behavior of skeletal and cardiac muscle observed in the ANT1-knockout animal model of oxidative stress . Moreover, MnSOD and CuZnSOD expression increases several fold in Saccharomyces cerevisiae during menadione-induced oxidative stress, without a parallel increase in their activities . CuZnSOD inactivation has been ascribed to hydrogen peroxides , whereas inactivation of MnSOD relates to peroxynitrite species, generated by the interaction of superoxide anion with nitric oxide , which can nitrate critical tyrosine residues causing loss of enzyme activity . Experiments are currently in progress in our laboratory to evaluate protein nitrosylation in control and mutated cybrids cultured in glu-DMEM.
Overall, our results indicate that, in spite of low efficiency in some antioxidant enzymes, particularly evident in cells affected by the most severe LHON pathogenic mutations, the cybrids maintained in glucose-supplemented medium successfully manage the increased generation of ROS. This is indicated by GSH and GSSG concentrations, which are not significantly modified in comparison with those of controls, and by the normal cell growth .
Antioxidant defences in cybrids cultured in gal-DMEM
When the cells were cultured in glucose-free/galactose-supplemented medium, the situation dramatically changed. The replacement of glucose by galactose in the culture medium (gal-DMEM) forces the cells to rely on oxidative phosphorylation for ATP production, which in the case of LHON cybrids is severely impaired when driven by complex I substrates . Galactose medium induces a dramatic time-dependent depletion of cellular ATP content  and a wave of apoptotic cell death . As peroxide scavenging via pyruvate as well as via NADPH-dependent reactions is decreased in these conditions, because the restricted flow of galactose to glucose 6-phosphate decreases NADPH availability [20,32], incubation in galactose is expected to induce a metabolic/oxidative stress crisis, which aggravates the pathogenicity of LHON mutations. In fact, LHON cybrids incubated in gal-DMEM show dramatic modifications in their antioxidant defences. All LHON cybrids had GR activities that were significantly lower than in control cybrids, with a particularly large decrease in the 11778/ND1 mutated cells. Large modifications in glutathione homeostasis were also evident in cybrids carrying 11778/ND4 or 3460/ND1, as reflected by their earlier and more marked increases in GSSG concentration compared with the control and 14484/ND6 mutated cybrids. This accumulation of GSSG is the likely result of increased ROS production, when this exceeds the metabolic capabilities of GPx and GR. A burst of oxidative stress resulting in enhanced formation of peroxynitrite  in LHON cybrids may also explain the significant decrease in MnSOD activity observed after a 24-h incubation in gal-DMEM.
In cybrids bearing the most severe 3460/ND1 and 11778/ND4 mutations, we found a time-dependent, large increase in GSH. This is not surprising, as very similar cellular behavior, i.e. concurrent increase in GSSG and GSH, has been observed in multidrug-resistant human breast carcinoma cells treated with a glucose-free medium . This indicates that the cells in galactose medium increase glutathione synthesis, in an attempt to counteract the increased production of intracellular pro-oxidants. As GR activity is greatly impaired in 3460/ND1 and 11778/ND4 mutated cybrids, and NADPH availability is decreased in gal-DMEM, the GSH increase observed in these cells may be due to de novo GSH synthesis, a two-step process catalyzed by γ-glutamylcysteine synthetase and GSH synthetase . We did not directly measure γ-glutamylcysteine synthetase expression and/or activity in cybrids in our experimental conditions. However, our hypothesis is supported by a study reporting that rat lung epithelial L2 cells respond to menadione-induced oxidative stress, with a 2.5-fold increase in their GSH content obtained through increased γ-glutamylcysteine synthetase activity  and increased transcription of the regulatory subunit of γ-glutamylcysteine synthetase itself . In spite of the increase in GSH, however, the cybrids harboring the most severe 3460/ND1 and 11778/ND4 mtDNA mutations failed to maintain the percentage of GSSG vs. (GSH + GSSG) at values similar to those of the controls or the cybrids with the 14484/ND6 mutation, indicating a situation of greater cellular distress. This imbalance in glutathione homeostasis may be closely connected with the apoptotic death of LHON cybrids grown in galactose medium . It is well known that the redox state of thiols regulates the mitochondrial permeability transition pore , its opening being responsible for energy uncoupling, diminished intracellular ATP concentrations, and release of cytochrome c and other pro-apoptotic factors.
Our study shows that cybrids carrying the three most common mtDNA point mutations associated with LHON show evidence of low efficiency of some of the antioxidant enzymes, probably because of post-translational events. The extent of this phenomenon seems to be related to the severity of the biochemical defect associated with the LHON mutation and possibly to the amount of ROS generated by mitochondria, correlating also with the clinical phenotype of the disease. Thus, the 14484/ND6 mutation, which is associated with a benign visual prognosis and normal complex I activity  and no significant decrease in oxygen consumption (present data), has the lowest ROS production  and the mildest impairment in antioxidant activity (present results). In contrast, the 3460/ND1 and the 11778/ND4 mutations, which consistently decrease complex I activity [3,6] and oxygen consumption (present data) and are associated with severe neuropathology , are associated with the highest mitochondrial ROS production  and the least efficient antioxidant defences (present results). However, while in glucose medium, all LHON-affected cybrids retain the ability to buffer their dysfunctions, as shown by the lack of modification in glutathione homeostasis and by their normal growth. However, the modifications described in the antioxidant machinery result in greater vulnerability of the cells to metabolic/oxidative stress, as seen in galactose medium. Under these conditions, a further decrease in some antioxidant activities (MnSOD and GR) and drastic impairment of glutathione homeostasis occur, particularly in cybrids with 3460/ND1 and 11778/ND4 mutations. Taken together these results provide further insight into the pathophysiological consequences of LHON mutations, indicating that the unstable balance in which LHON cells live may be upset by any exogenous or endogenous condition (e.g. exposure to drugs and/or toxins, or changing hormonal status) that favors ROS generation. This may precipitate the cells into a metabolic crisis which in vivo may lead to apoptotic death of retinal ganglion cells and clinically expressed LHON.
Tissue culture reagents were purchased from Gibco-Invitrogen (Milan, Italy). Cumene hydroperoxide, NADPH, GSH, GSSG, xanthine, xanthine oxidase (from buttermilk), glutathione reductase (from baker's yeast), nitroblue tetrazolium, 2-vinylpyridine and albumin were obtained from Sigma Chemical Co. (St Louis, MO, USA). NaCl/Pi from Oxoid had the following composition: NaCl 8 g·L−1, KCl 0.2 g·L−1, Na2HPO4 1.15 g·L−1 and KH2PO4 0.2 g·L−1 (pH 7.3). All other reagents were of analytical grade and were used as received.
Construction and characterization of cybrid cell lines
Cybrid cell lines were constructed from fibroblasts obtained, after informed consent, from skin biopsies of six members of five unrelated families with clinically and molecularly defined LHON, previously reported [4,5], and from five unrelated controls. Cells, grown in DMEM supplemented with 10% fetal calf serum, were then enucleated and fused with osteosarcoma-derived mtDNA-less cells (rho°206 derived from 143B.TK–, a gift from G. Attardi) as described . After selection in uridine-deprived bromodeoxyuridine-supplemented medium, several cybrid clones (a minimum of two for each individual) repopulated with mtDNA from each fibroblast cell line were isolated and expanded. Moreover, to avoid possible confounding effects due to different functional profiles of different batches of 143B.Tk–, and to indirectly check the unstable nuclear genome of the 143B cells, we measured the respiratory capacity of 143B cells frozen just after being brought from the donor laboratory (1993) or after various periods of time in culture (frozen in 1996, 1999, 2002). The observed values were all within the range of experimental variation (± 10%).
The cybrids have been regularly checked for the presence of the LHON pathogenic mutations 3460/ND1, 11778/ND4 and 14484/ND6 following the standard PCR/restriction fragment length polymorphism method [16,17]. All LHON cybrids used carried stably homoplasmic mtDNA mutations, reconfirmed every 3 to 5 months.
Moreover, a functional check of the cybrids was carried out by determination of oxygen consumption, as described previously . Briefly, the rate of oxygen consumption was measured in intact cells with a Gilson 5/6 oxygraph on samples of (4–5) × 106 cells in 1.85 mL DMEM lacking glucose supplemented with 5% dialyzed fetal calf serum at 37 °C. By this method, we tested five control lines, three lines with the 11778/ND4 mutation, two lines with 3460/ND1 and two with 14484/ND6 mutations. Each line is the result of a cybridization from a different individual, and each bar shown in the graph of Fig. 1 is the result of averaging data obtained from two to seven different clones each assessed in at least three independent experiments.
A representative clone of each independent line (HPC7: control; HFF3: 11778/ND4; HMM12: 3460/ND1; HL180: 14484/ND6) was used for the extensive study detailed in the following sections.
Cybrid cell lines were grown in DMEM supplemented with 10% fetal calf serum, 2 mm l-glutamine, 100 U·mL−1 penicillin, 100 µg·mL−1 streptomycin and 0.1 mg·mL−1 bromodeoxyuridine. For the experiments, the cells (≈ 1.8 × 106) were plated on 10-cm Petri dishes in 8 mL of the above reported DMEM and maintained at 37 °C in an incubator under a humidified 5% CO2 atmosphere. After 24 h, the culture medium was replaced with fresh complete DMEM supplemented with 25 mm glucose (glu-DMEM) or with glucose-free DMEM supplemented with 5 mm galactose, 5 mm sodium pyruvate and 5% fetal calf serum (gal-DMEM), as described by Ghelli et al. . The cells were incubated at 37 °C for 24 more hours.
Measurement of GSH and GSSG
Cellular GSH and GSSG concentrations were measured enzymatically , as described previously . Briefly, 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 into 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 [≈ (5–6) × 106 for each determination] were washed once with NaCl/Pi and then treated with 6% metaphosphoric acid (1 mL per dish) at room temperature. After 10 min, the acid extract was collected, centrifuged for 5 min at 18000 g at 4 °C, and processed. The cellular debris remaining on the plate was solubilized with 0.5 m KOH and assayed for protein content as described by Lowry et al. . For total glutathione determination, the above acid extract was diluted (1 : 6) in 6% metaphosphoric acid; thereafter to 0.1 mL supernatant were added 0.75 mL 0.1 m potassium phosphate/5 mm EDTA buffer (pH 7.4), 0.05 mL 10 mm 5,5′-dithiobis-(2-nitrobenzoic acid) (prepared in 0.1 m phosphate buffer) and 0.08 mL 5 mm NADPH. After a 3-min equilibration period at 25 °C, the reaction was started by the addition of 2 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 3 min 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 (0.05 to 0.4 µg·mL−1) of GSH against the rate of change in A412. GSH standards were prepared daily in 6% metaphosphoric acid 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-vinylpyridine at room temperature for 60 min. In a typical experiment, 0.15 mL supernatant was treated with 3 µL undiluted 2-vinylpyridine. Then 9 µL triethanolamine was added. The mixture was vigorously mixed, and the pH was checked; it was generally between 6 and 7. After 60 min, 0.1 mL aliquots of the samples were assayed by 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 (0.05 to 0.20 µg·mL−1) against the rate of change in absorbance.
GSH present in the samples was calculated as the difference between total glutathione and GSSG concentrations.
Assay of antioxidant enzyme activities
For measurement of GPx, GR and catalase activities, monolayer cells [≈ (2–3) × 106] were washed three times with NaCl/Pi before treatment directly on the dish with a solution consisting of 0.25 m sucrose, 10 mm Tris/HCl (pH 7.5), 1 mm EDTA, 0.5 mm phenylmethanesulfonyl fluoride, 0.5 mm 1,4-dithio-dl-threitol and 0.1% (v/v) Nonidet (solution A), to obtain complete lysis of intracellular organelles. Cells were then scraped from the plate, and the samples were centrifuged for 30 min at 105 000 g. Protein measurements  and enzyme assays were carried out on the clear supernatant fractions.
Total GPx activity was measured by the coupled enzyme procedure with glutathione reductase, as described by Prohaska & Ganther , using cumene hydroperoxide as substrate. Enzyme activity was monitored by following the disappearance of NADPH at 340 nm for 3 min at 25 °C. The incubation medium (final volume 1 mL) had the following composition: 50 mm KH2PO4 (pH 7.0), 3 mm EDTA, 1 mm KCN, 1 mm GSH, 0.1 mm NADPH, 2 U glutathione reductase and ≈ 300 µg protein. After a 3-min 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 a molar absorption coefficient obtained from a standard curve of NADPH (0.02 to 0.1 µmol·mL−1), and GPx activity was expressed in nmol NADPH consumed per mg protein·min−1.
GR activity was measured by the method of Carlberg & Mannervik , by following the rate of oxidation of NADPH by GSSG at 340 nm for 3 min at 25 °C. The reaction mixture (final volume 1 mL) contained: 0.1 m KH2PO4 (pH 7.6), 0.5 mm EDTA, 1 mm GSSG, 0.1 mm NADPH, and ≈ 300 µg protein. The specific activity was calculated by using a molar absorption coefficient obtained from a standard curve of NADPH (0.02 to 0.1 µmol·mL−1), and GR activity was expressed in nmol NADPH consumed per mg protein·min−1.
Total catalase activity was assayed by the method of Aebi . 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 1 mL) consisting of 50 mm phosphate buffer (pH 7.0) and ≈ 100 µg protein. Catalase activity was expressed as U per mg protein, assuming that 1 U catalase decomposes 1 µmol H2O2·min−1.
For MnSOD and CuZnSOD assays, after treatment of 2 × 106 cells with solution A, an aliquot (0.6 mL) of cell lysate was sonicated, on ice (2 × 30 s bursts) with a Labsonic U2000 sonicator (B. Braun Biotech International, Melsungen, Germany) and then centrifuged for 30 min at 105 000 g as described by Siemankowski et al. . The supernatant was collected and dialyzed overnight in cold double-distilled water to remove small interfering substances. Enzyme assays were carried out by the method of Oberley & Spitz , with minor modifications. Briefly, in 1 mL medium consisting of 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 µm nitroblue tetrazolium to monitor superoxide formation by following the changes in colorimetric absorbance at 560 nm for 5 min at 25 °C. The catalytic activities of the samples were evaluated as their ability to inhibit the rate of nitroblue tetrazolium reduction; increasing amounts of protein (5 to 150 µg) were added to each sample until maximum inhibition was obtained. SOD activity was expressed as U per mg protein, 1 U SOD activity being defined as the amount of protein causing half-maximal inhibition of the rate of nitroblue tetrazolium reduction. To assess MnSOD activity, cell fractions were preincubated for 60 min at 0 °C in the presence of 5 mm KCN, which produces total inhibition of Cu/ZnSOD. The latter activity was calculated as the difference between activities in the absence and presence of KCN.
Western blot assay of CuZnSOD and MnSOD
On the same cybrid cell lysates, obtained as reported above and used for SOD activity assays, Western blotting was carried as described by Dieterich et al. . Equal amounts of protein (40 µg per lane) denatured in electrophoresis buffer containing 100 mm Tris/HCl (pH 6.8), 8 mm dithiothreitol, 2% SDS, 2% glycerol, and 0.05% bromophenol blue at 95 °C were electrophoresed on SDS/polyacrylamide gel (10% gel) and electrotransferred to a poly(vinylidene difluoride) membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in 0.02 m Tris/HCl buffer, pH 7.5, containing 0.137 m NaCl and 0.1% (v/v) Tween 20 (TBST) for 1 h at room temperature and then incubated with sheep anti-(human CuZnSOD) IgG (Calbiochem, Darmstadt, Germany) diluted 1 : 1000, anti-(human MnSOD) IgG diluted 1 : 10 000 (gift from John Guy, University of Florida), or anti-(human tubulin) IgG (Sigma) diluted 1 : 1000. After being washed with NaCl/Pi/0.1%Tween, the blots were incubated with horseradish peroxidase-conjugated anti-sheep IgG (Calbiochem) diluted 1 : 10 000 at room temperature for 1 h. After a final wash of the membrane with TBST, the immunoreactivity was detected with enhanced chemiluminescence detection solution (Sigma). The immunoreactivity was detected in the same way as described above. Quantitative analysis of the blots was carried out using the image j software (http//rsb.info.nih.gov/ij/).
Results are expressed as arithmetic means ± SD. Comparisons were made by one-way analysis of variance. A P value of less than 0.05 was considered significant.
This work was supported, in part, by grants from MIUR (Italy). The financial support of Telethon Italy (project No. GGP02323 to VC and AM) is gratefully acknowledged.