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Increased expression of manganese-superoxide dismutase in fibroblasts of patients with CPEO syndrome
Molecular Genetics and Metabolism 80 (2003) 321–329 www.elsevier.com/locate/ymgme
Increased expression of manganese-superoxide dismutase in fibroblasts of patients with CPEO syndrome Ching-You Lu,a Edward K. Wang,b Hsin-Chen Lee,c Huey-Jen Tsay,d and Yau-Huei Weia,* a
Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC b Section of Peripheral Neurology, Taipei Veterans General Hospital, Taipei 112, Taiwan, ROC c Department of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan, ROC d Institute of Neuroscience, National Yang-Ming University, Taipei 112, Taiwan, ROC Received 22 May 2003; received in revised form 31 July 2003; accepted 4 August 2003
Abstract Alterations in the expression of free radical scavenging enzymes and production of reactive oxygen species (ROS) in tissue cells may contribute to the pathogenesis of mitochondrial diseases such as chronic progressive external ophthalmoplegia (CPEO) syndrome. Since the mitochondria with impaired respiratory function in affected tissues generate more ROS via electron leakage, we examined the expression levels of free radical scavenging enzymes in primary culture of muscle fibroblasts of eight patients with CPEO syndrome. The results showed that the enzyme activity and protein levels of Mn-SOD of the fibroblasts from CPEO patients were significantly increased but those of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) were not increased compared with controls. A similar pattern was observed in the mRNA levels of Mn-SOD and GPx in muscle fibroblasts of all CPEO patients. The activity ratios of Mn-SOD/catalase and Mn-SOD/GPx in muscle fibroblasts of the CPEO patients were increased 1.7–3.4 and 1.8- to 5.3-fold, respectively, compared to those of the controls. Moreover, by using flow cytometry we found that the production of O 2 and H2 O2 in the fibroblasts was about 2 times higher than those of controls. The 8-OHdG/dG ratios in total DNA of muscle biopsies from three CPEO patients were much higher than those of age-matched controls as determined by high performance liquid chromatography (HPLC). In the light of these findings, we suggest that the increase in expression of Mn-SOD, ROS production and oxidative damage in affected tissues may play an important role in the pathogenesis and progression of the CPEO syndrome. Ó 2003 Elsevier Inc. All rights reserved. Keywords: CPEO syndrome; Mitochondrial DNA; Free radical scavenging enzyme; Mn-superoxide dismutase; Oxidative stress; Muscle fibroblasts
Introduction Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial encephalomyopathies, which is characterized by myopathy, progressive limitation of eye movement due to weakness of the extraocular muscle [1]. The disease can be either sporadic [2] or transmitted through maternal lineage [3] or of autosomal dominant inheritance [4]. Many of these symptoms are associated with ragged-red fibers in muscle, which are formed by the accumulation of structurally abnormal mitochondria with paracrystalline inclusions [1]. Two types of mutation in mitochondrial
* Corresponding author. Fax: +886-2-2826-4843. E-mail address: [email protected] (Y.-H. Wei).
1096-7192/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2003.08.005
DNA (mtDNA) have been found in muscle of CPEO patients. Most of the patients with CPEO harbor largescale deletions of mtDNA and a portion of the remaining CPEO patients harbor the A3243G mutation in the tRNALeuðUURÞ gene, which is more commonly associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) [1,5]. Recently, several nuclear genes have been identified to associate with dominant and recessive forms of CPEO characterized by accumulation of large-scale mtDNA deletions, including the genes coding for adenine nucleotide translocator 1 (ANT1) at locus 4q34–35 [6], a putative mitochondrial helicase (Twinkle) at locus 10q24 [7], and the mitochondrial DNA polymerase c at locus 15q22–26 [8]. Mitochondrial respiratory chain dysfunction in affected tissues of patients with mitochondrial diseases not
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only results in decreased energy production, but also causes increased generation of reactive oxygen species (ROS) with consequent oxidative stress and cellular damage [5]. Increased lipid peroxidation and reduced glutathione (GSH) level have been observed in blood plasma and erythrocytes of patients with CPEO syndrome [9]. Moreover, it has been demonstrated that the specific contents of 8-OHdG and lipid peroxides are significantly increased in cybrids harboring a high level of the 4977 bp-deleted mtDNA as compared with those of the control cybrids [10]. The ratio of glutathione disulfide/reduced glutathione (GSSG/GSH) and lipid peroxide content in the cybrids harboring >95% A3243G mutant mtDNA were significantly higher than those of control cybrids containing undetectable mutant mtDNA [11]. These findings have provided strong support to the notion that increased oxidative damage to vital biomolecules in affected tissues of CPEO patients is a result of the elevation of intracellular oxidative stress elicited by mitochondrial dysfunction in the target cells harboring mtDNA mutations [5]. To cope with the free radicals and ROS by-products generated from the mitochondrial respiratory chain, human cells have developed an antioxidant defense system consisting of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and an array of low-molecular-weight antioxidants including ascorbic acid, a-tocopherol, and GSH. Mn-SOD, located in the mitochondria, and Cu,Zn-SOD, located in the cytoplasm, catalyze the dismutation of superoxide anions into hydrogen peroxide and molecular oxygen. Then, GPx uses reduced glutathione to reduce hydrogen peroxide or CAT converts two molecules of hydrogen peroxide into water and oxygen. A fine balance among the free radical scavenging enzymes is necessary to prevent the oxidative damage to vital biomolecules in tissue cells. Recently, increased immuno-staining of MnSOD and Cu,Zn-SOD has been reported in cytochrome c oxidase (COX)-deficient muscle fibers in patients with
mtDNA mutations and respiratory chain defects [12]. Moreover, it was reported that the strong immunoreactivity to the Mn-SOD and Cu,Zn-SOD in deficient fibers correlated well with the presence of apoptotic nuclei [13]. These observations suggest that over-expression of Mn-SOD and Cu,Zn-SOD alone is not sufficient to dispose of increased ROS and may even induce or promote apoptotic cell death in fibers with defective respiratory function. We therefore hypothesized that alterations in the expression of free radical scavenging enzymes or increased intracellular level of ROS in affected tissues may contribute to the pathogenesis of mitochondrial disease such as CPEO syndrome. To test this hypothesis, we cultured muscle fibroblasts from eight patients with CPEO syndrome and examined the enzyme activities and mRNA levels of Mn-SOD, CAT, and GPx. The 8-OHdG content in the fibroblasts was also measured as an index of oxidative damage to DNA. We found an increase in Mn-SOD expression, ROS production, and oxidative DNA damage in muscle fibroblasts of these CPEO patients. The results obtained in this study have provided evidence to support the hypothesis that endogenous oxidative stress elicited by imbalanced expression of free radical scavenging enzymes in affected tissues of the patients may play a role in the pathogenesis and progression of CPEO syndrome.
Materials and methods Patients Muscle biopsies were procured from eight patients with CPEO syndrome and control subjects with their consent by one of the authors (E. K. Wang) with the assistance of attending physicians at Taipei Veterans General Hospital. The clinical features and laboratory findings of the CPEO patients are summarized in Table 1. The most common clinical symptoms of these
Table 1 Clinical features of the patients with CPEO syndrome examined in this study Clinical features
Patients 1
2
3
4
5
6
7
8
Age Sex Age of onset Ptosis External ophthalmoplegia Muscle biopsies ragged-red fibers MtDNA mutation in muscle fibroblasts Large-scale deletion Size (bp) A3243G point mutation Proportion of the mutated mtDNA (%) in fibroblasts
46 F 34 + + + +
46 F 26 + + + +
61 M 50 + + + +
55 F 45 + + + )
63 M 56 + + + )
50 F 30 + + + +
26 F 18 + + + )
26 F 15 + + + +
4977 ) 6.5
NA ) NA
4977 ) 4.5
+ 0.75
+ 0.75
4366 ) 20
+ 16
6190 ) 12
NA: Not available.
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patients were external ophthalmoplegia, ptosis, muscle weakness, and exercise intolerance. All of the 8 CPEO patients had ragged-red fibers in skeletal muscle upon staining with modified Gomori trichrome. Electron micrographs revealed large accumulations of abnormal mitochondria in the subsarcolemmal regions of the muscle fibers of these patients. Cell lines and culture condition Primary cultures of fibroblasts were established from muscle biopsies of patients with CPEO syndrome and healthy subjects of different ages. The muscle fibroblasts were grown in DulbeccoÕs modified EagleÕs medium (DMEM, Gibco-BRL, Bethesda, MD) with the supplement of 15% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 10 lg/ml pyruvate, and 5 lg/ml uridine. The cells were grown to confluence before use in a 150 cm2 petri dish in a growth chamber set at 37 °C under humidified atmosphere of 5% CO2 and 95% air. Assay of the activities of free radical scavenging enzymes To determine the enzyme activities of Cu,Zn-SOD, Mn-SOD, CAT, and GPx, muscle fibroblasts were trypsinized and washed with ice-cold phosphate-buffered saline (PBS, pH 7.3) and the cell pellets were collected by centrifugation. The cells were suspended in a pre-cooled NEP buffer (150 mM NaCl, 0.5 mM EDTA, and 100 mM phosphate buffer, pH 7.5) containing 0.25% sodium cholate and protease inhibitors (Boehringer Mannheim, Germany). The cell suspension was then mixed with an equal volume of glass beads and vortex-mixed vigorously for 8 intervals of 10 s per round, separated by 30 s cooling intervals. The cell lysate was spun down at 800g for 5 min, and the supernatant was collected for the assay of the activities of free radical scavenging enzymes. CAT activity was determined by monitoring the rate of decomposition of H2 O2 from the decrease in absorbance at 240 nm. GPx activity was determined using a coupled assay in which enzyme activity is proportional to the rate of NADPH oxidation as described by Lu et al. [14]. Total SOD activity was assayed by monitoring nitroblue tetrazolium (NBT) reduction according to Spitz and Oberley [15] with some modifications. The inhibition by SOD of NBT reduction by O 2 in the aerobic xanthine/xanthine oxidase system was followed by change of absorbance at 560 nm. One unit of SOD is defined as the amount of enzyme that causes 50% inhibition of NBT reduction under the assay condition described. Mn-SOD activity was differentiated from Cu,Zn-SOD by its resistance to NaCN. In the absence of NaCN, total SOD activity was measured and the MnSOD activity was assayed by monitoring NBT reduction in the presence of 5 mM NaCN.
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Determination of protein levels of free radical scavenging enzymes The cell lysate obtained from above-mentioned preparation was subjected to Western blot analysis of the protein levels of free radical scavenging enzymes. An aliquot of 25 lg of the cell lysate was subjected to 10% SDS–PAGE and transferred onto a Hybond nitrocellulose membrane (Amersham–Pharmacia Biotech, Little Chalfont, Buckinghamshire, England). After blocking for 1 h with 5% dried fat-free milk in Tris-buffered saline-Tween (TBST) buffer (20 mM Tris–HCl, pH 7.6, 0.9% NaCl, and 0.2% Tween 20), the membrane was incubated for 2 h with the primary antibody at room temperature. After washing three times with TBST, the blot was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then an enhanced chemiluminescence detection kit (Amersham–Pharmacia Biotech) was used. Autoradiograph of the protein signals in the Western blot was obtained by exposure of a Kodak X-ray film. The relative protein content of each free radical scavenging enzyme was normalized with that of GAPDH. Mouse anti-GAPDH and mouse anti-Mn-SOD monoclonal antibody were purchased from CHEMICON international (Temecula, CA), and sheep anti-human Cu, Zn-SOD antibody was bought from Biodesign International (Saco, Maine). RNA isolation and Northern hybridization Total RNA was isolated from muscle fibroblasts cultured under the above-mentioned condition [16]. Each RNA sample (30 lg) was dissolved in 1 ll of 10 MOPS running buffer, 3.5 ll of 12.3 M formaldehyde, and 10 ll deionized formamide and then denatured by incubation at 55 °C for 30 min. The RNA was electrophoresed on a 1.0% agarose denaturing gel and blotted overnight onto a nylon membrane in a 6 SSC buffer. The RNA blots were UV cross-linked and immersed completely in the 6 SSC buffer and prehybridized at 65 °C for 15 min in the rapid hybridization buffer. Purified cDNAs were labeled with [a-32 P]dCTP by random priming using a ready prime kit (Amersham Bioscience, Buckinghamshire, UK). Hybridization was performed at 65 °C for 2 h in the rapid hybridization buffer plus the cDNA probe of either Mn-SOD, CAT or GPx and washed with 2 SSC buffer containing 0.1% SDS at room temperature for 15 min. The blots were then washed twice with 0.1 SSC buffer/0.1% SDS at 65 °C for 15 min. Autoradiographs were obtained by exposure for 24–30 h of a Kodak X-ray film with an intensifying screen at )70 °C. The amount of the mRNA of each free radical scavenging enzyme was calculated on the basis of the intensity of its band in the RNA blot normalized by simultaneous hybridization with a mouse
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muscle b-actin cDNA probe. The cDNA probes of human Mn-SOD (546 bp), CAT (919 bp), and GPx (478 bp) were cloned by PCR using the published cDNA sequences of the respective genes, and the b-actin cDNA probe was purchased from Stratagene (La Jolla, CA). Flow cytometric measurement of superoxide anion and hydrogen peroxide The intracellular level of superoxide anions was measured by flow cytometry using hydroethidine (HE; Molecular Porbes, Eugene, OR). HE is a non-fluorescent compound that can diffuse through cell membranes, which then reacts rapidly with O 2 to form the red fluorescent product of ethidium. On the other hand, the intracellular level of H2 O2 was measured by use of the fluorescent dye dihydro-20 ,70 -dichlorofluorescin diacetate (H2 DCF-DA; Molecular Probes), which is converted to green fluorescent 20 ,70 -dichlorofluorescein (DCF) after reacting with H2 O2 . Usually, HE or DCFH-DA was added to the culture dish containing approximately 1 106 cells in DMEM (without phenol red). After 15 min of incubation at 37 °C, cells were kept on ice for immediate analysis on a FACScan flow cytometer (Becton–Dickinson, Bedford, MA) equipped with a 488 nm argon laser. The excitation wavelength was set at 488 nm. The observation wavelength of 530 nm was chosen for DCF green fluorescence and 585 nm for ethidium red fluorescence and the intensities of emitted fluorescence were collected on FL1 and FL2 channels, respectively. Data were acquired and analyzed using the Cell Quest software (Becton–Dickinson).
Analysis of 8-OHdG content in total DNA from muscle tissues The relative amount of 8-OHdG in total DNA of muscle biopsies from CPEO patients and control subjects was determined by the method developed by Shigenaga et al. [17] with modifications as described previously by Lu et al. [14].
Results Morphological characteristics of the muscle fibroblasts of CPEO patients The growth kinetics and morphological characteristics of the muscle fibroblasts of CPEO patients were quite different from those of the muscle fibroblasts of controls. It took 2 weeks for the muscle fibroblasts of controls to grow to confluence after subculture at 1:3 split. But at least 3 weeks were required for the muscle fibroblasts of our CPEO patients to grow to confluence after 1:3 split at subculture. Moreover, the muscle fibroblasts of all the CPEO patients examined showed significantly larger size and more flattened and extended morphology compared with the muscle fibroblasts of controls upon examination by a light microscope. The maximal number of muscle fibroblasts from CPEO patients was less than that of muscle fibroblasts of controls. On the other hand, muscle fibroblasts of the CPEO patients exhibited many more vacuoles and granular particles, which were clustered near the area surrounding the nucleus (Fig. 1).
Fig. 1. Morphology of muscle fibroblasts from CPEO patients and healthy subjects. A represents normal muscle fibroblasts. B–F show the muscle fibroblasts from patients CPEO2, CPEO3, CPEO4, CPEO5, and CPEO6 at a magnification of 150.
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MtDNA mutation in the muscle fibroblasts of CPEO patients Molecular analysis showed that mtDNA deletions of different sizes existed in muscle fibroblasts of patients CPEO1, CPEO2, CPEO3, CPEO6, and CPEO8 (Table 1). On the other hand, CPEO4, CPEO5, and CPEO7 patients harbored the A3243G point mutation in the mitochondrial tRNALeuðUURÞ gene. It should be noted that although the muscle biopsies from CPEO patients usually had high proportion of mutant mtDNA (above 60%), the proportion of mutant mtDNA was much lower in cultured muscle fibroblasts as compared to that of the corresponding muscle biopsies. A similar decline in the proportion of mtDNA with A3243G mutation was previously observed in cultured muscle fibroblasts compared with muscle biopsies [18]. Activities of free radical scavenging enzymes in muscle fibroblasts The enzyme activities of Cu,Zn-SOD, Mn-SOD, CAT, and GPx in muscle fibroblasts from the eight CPEO patients are summarized in Table 2. The activities of Mn-SOD in muscle fibroblasts of the CPEO patients were approximately 2-fold higher compared with those of controls. By contrast, the activities of Cu,Zn-SOD, CAT or GPx did not show much changes. Upon further analysis, we found that the activity ratios of Mn-SOD/ CAT and Mn-SOD/GPx in muscle fibroblasts from CPEO patients were significantly higher (p < 0:01) than the corresponding values of the controls (Figs. 2A and B). The ratios of enzyme activity between the mutant and control fibroblasts were 1.7–3.4 for Mn-SOD/CAT and 1.8–5.3 for Mn-SOD/GPx, respectively.
Fig. 2. The enzyme activity ratios of Mn-SOD/catalase (A) and MnSOD/103 GPx (B) of CPEO patients and controls. *Indicates significant difference compared with those of the control muscle fibroblasts, p < 0:01. The box-whisker plot made by using the Sigma Plot software extended from the 25th to the 75th percentile of interquartile range (IQR). The centerline indicates the median of enzyme activity ratio of Mn-SOD/catalase or Mn-SOD/GPx in CPEO patients and controls. The whiskers at the end of each box are defined as the 75th percentile of +1.5 IQR and the 25th percentile of –1.5 IQR. The black dots shown on the two plots represent the activity ratios outside the 1.5 IQR.
Protein levels of free radical scavenging enzymes in muscle fibroblasts The enzyme protein levels assessed by Western blotting were found to change in parallel with the change of the activities of free radical scavenging enzymes in the fibroblasts (Fig. 3). The Mn-SOD protein level in fibroblasts of the seven CPEO patients were 3- to 6-fold higher than those of the healthy subjects, but the protein
Fig. 3. Protein levels of different antioxidant enzymes from muscle fibroblasts of CPEO patients. GAPDH was used as internal standard in this Western blot analysis. Lanes 1 and 2 represent muscle fibroblasts from healthy subjects. Lanes 3–9 indicate muscle fibroblasts from patients CPEO1, CPEO2, CPEO4, CPEO5, CPEO6, CPEO7, and CPEO8, respectively.
Table 2 Comparison of the activities of free radical scavenging enzymes in muscle fibroblasts from the patients with CPEO syndrome with those of agematched controls Subject
Cu,Zn-SOD (unit/mg)
Mn-SOD (unit/mg)
Catalase (103 unit/g)
GPx (unit/g)
CPEO (n ¼ 8) Control (n ¼ 5)
149 68 118 24
260 24 116 34
6.2 1.3 6.6 1.6
56.0 15.0 65.9 6.7
The enzyme activities are expressed as means SD. * Significantly different from muscle fibroblasts of controls, p < 0:001, using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined were primary cultures between three and six passages.
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levels of Cu,Zn-SOD did not show any significant change. RNA levels of free radical scavenging enzymes in muscle fibroblasts To examine gene expression of Mn-SOD, CAT, and GPx in muscle fibroblasts from the CPEO patients and controls, we measured the mRNA levels of these antioxidant enzymes. The Northern blot analysis of the expression of these free radical scavenging enzymes is shown in Fig. 4. The mRNA levels of the enzymes were determined by densitometric scanning of the autoradiographs of Northern blots. The results are expressed in arbitrary units after normalization with the
intensity of the b-actin mRNA band. The mRNA levels of Mn-SOD, CAT, and GPx in muscle fibroblasts of the CPEO patients are summarized in Table 3. The changes in mRNA levels of Mn-SOD and GPx were similar to those observed for the activities and protein levels of the corresponding enzymes. In contrast to the activity, the mRNA level of CAT was also increased in muscle fibroblasts of CPEO patients. The mRNA levels of Mn-SOD and CAT in muscle fibro-
RNA ratio of Mn-SOD/CAT
326
4
A A
#
3
2
1
0
RNA ratio of Mn-SOD/GPx
2
B B
# 1
0
Control Fig. 4. The mRNA levels of Mn-SOD, catalase, and GPx in muscle fibroblasts of the patients with CPEO syndrome. Total RNA was extracted and analyzed by Northern blot and hybridized with 32 P-labeled cDNA probes of Mn-SOD, catalase, GPx, and b-actin as described in Materials and methods. The b-actin mRNA was used as an internal standard for normalizing the amount of RNA in each lane. The intensity of each band was expressed in arbitrary scale because it was determined by different duration of exposure of the autoradiographs and by cDNA probes of different size. Lanes 1–8 indicate the mRNA levels of muscle fibroblasts from the eight CPEO patients, and lanes 9 and 10 show the mRNA levels of Mn-SOD of the muscle fibroblasts from two healthy subjects, respectively.
CPEO
Fig. 5. The mRNA ratios of Mn-SOD/catalase (A) and Mn-SOD/GPx (B) in muscle fibroblasts of CPEO patients and controls. # Indicates significant difference compared with those of the control fibroblasts, p < 0:01. The box-whisker plots made by using the Sigma Plot software extended from the 25th to the 75th percentile of interquartile range (IQR). The centerline indicates the median of the activity ratio Mn-SOD/catalase or Mn-SOD/GPx in CPEO patients and controls. The whiskers at the end of each box are defined as the 75th percentile of +1.5 IQR and the 25th percentile of )1.5 IQR. The black dots shown on the two plots represent the activity ratios outside the 1.5 IQR.
Table 3 Comparison of the mRNA levels of Mn-SOD, catalase and glutathione peroxidase of muscle fibroblasts of patients with CPEO syndrome with those of age-matched controls Subject
Mn-SOD
Catalase
GPx
(Intensity ratio relative to that of b-actin) CPEO (n ¼ 8) Control (n ¼ 5)
0.68 0.17 0.22 0.06
0.34 0.07 0.16 0.04
0.69 0.20 0.72 0.09
Values for mRNAs of free radical scavengers Mn-SOD, catalase and GPx were normalized with the level of b-actin RNA. The data are presented as means SD. * Significantly (p < 0:001) compared with muscle fibroblasts of controls using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined were primary cultures between three and six passages.
C.-Y. Lu et al. / Molecular Genetics and Metabolism 80 (2003) 321–329
blasts of CPEO patients were approximately 3- and 2-fold, respectively, higher than those in the muscle fibroblasts of controls. We then examined the ratios of the mRNA level between Mn-SOD and CAT or between Mn-SOD and GPx. The ratios of mRNA level of Mn-SOD/CAT in the muscle fibroblasts of CPEO patients were higher than those of the controls but the difference did not reach statistical significance (Fig. 5A). The value was between 1.3 and 3.3 for CPEO patients compared with 1.38 in the controls. On the other hand, the mRNA ratio of Mn-SOD/GPx in the muscle fibroblasts of CPEO patients was increased 2- to 4-fold as compared with those of the controls (Fig. 5B). Intracellular levels of superoxide anions and hydrogen peroxide The contents of superoxide anions and hydrogen peroxide in muscle fibroblasts from the CPEO patients are listed in Table 4. We demonstrated that the muscle fibroblasts of CPEO patients exhibited a higher intracellular level of superoxide anions and hydrogen peroxide compared with muscle fibroblasts of controls.
Table 4 The intracellular levels of superoxide anions and hydrogen peroxide in muscle fibroblasts of patients with CPEO syndrome Subject
Superoxide anions (Ethidium intensity)
Hydrogen peroxide (DCF intensity)
CPEO (n ¼ 8) Control (n ¼ 5)
186 60
235 82
116 21
126 21
The intracellular contents of superoxide anions and hydrogen peroxide in muscle fibroblasts of patients with CPEO syndrome were measured by staining with hydroethidine and dihydro-20 ,70 -dichlorofluorescin diacetate and then analyzing by flow cytometry. The contents of superoxide anions and hydrogen peroxide are presented as means SD. * Significantly different from muscle fibroblasts of controls, p < 0:01, using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined in this study were primary cultures between three and six passages.
Table 5 Comparison of the 8-OHdG content in total cellular DNA of muscle biopsies of CPEO patients and controls Subject
8-OHdG/106 dG ratio
CPEO (n ¼ 3) Control (n ¼ 7)
50.4 23.8 5.8 1.6
* Significantly different from the controls, p < 0:05, using the StudentÕs t-test. Control data were obtained from 7 subjects receiving surgical operations who had none of the known mitochondrial diseases with ages between 40 and 70 years old.
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The 8-OHdG content in skeletal muscle of CPEO patients The oxidative damage to DNA in muscle biopsies of CPEO patients was found to be more severe than that of controls as indicated by the significant increase in the level of 8-OHdG/dG. As shown in Table 5, the levels of 8-OHdG/dG in muscle biopsies of CPEO patients were significantly higher than those of the controls.
Discussion In this study, we demonstrated for the first time that Mn-SOD was up-regulated at RNA, protein, and activity levels in muscle fibroblasts from CPEO patients. The activity and protein levels of Mn-SOD, but not those of Cu,Zn-SOD, CAT, and GPx, in muscle fibroblasts of the patients with CPEO syndrome were significantly higher than those of the controls. Moreover, Mn-SOD mRNA levels in muscle fibroblasts of CPEO patients were also significantly higher than those of the controls. The observation of a concurrent increase in gene expression and enzyme activity of Mn-SOD in muscle fibroblasts of CPEO patients is in agreement with previous reports on other mitochondrial disorders [19–22]. It has been reported that the severity of Complex I enzyme deficiency correlated with the increase of production of O 2 and the induction of gene expression of Mn-SOD [19]. In addition, it has been reported that the expression of MnSOD, but not of Cu,Zn-SOD, is increased in the skeletal muscle with ragged-red fibers in a number of patients with mitochondrial encephalomyopathies [20,21]. What causes the induction of Mn-SOD and whether there is a correlation with the severity of the disease have remained unclear. The elevation in gene expression and activity of Mn-SOD in muscle fibroblasts of the CPEO patients may be one of the responses of the cells to cope with overproduction of free radicals. Recently, Filosto et al. [21] conducted an immunohistochemical study on muscle biopsies from patients with mitochondrial diseases. They showed that the induction of gene expression of antioxidant enzymes correlates with the extent of mitochondrial proliferation. In addition, we have recently demonstrated that enhanced oxidative stress can lead to an increase in mitochondrial mass and mtDNA content of human lung fibroblasts [23]. These observations suggest a possible mechanism by which the elevated ROS production from defective electron transport chain results in enhanced oxidative stress, which subsequently causes mitochondrial proliferation and overexpression of the mitochondrial Mn-SOD. Indeed, we found that muscle fibroblasts from CPEO patients had about 1.6- and 1.9-fold higher levels of O 2 and H2 O2 as reported by HE and H2 DCF-DA, respectively. In addition, the oxidative damage to DNA, as indicated by the 8-OHdG/106 dG level, in muscle of CPEO patients
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was much more extensive than that in muscle of controls or patients with other types of diseases that are not associated with mtDNA mutation. Patients with MELAS or CPEO syndromes harboring A3243G mutation of mtDNA manifest very different clinical features. Thus, MELAS patients rarely present with ptosis and patients with CPEO syndrome never exhibited stroke-like episodes. We found that the activities and protein levels of free radical scavenging enzymes in muscle fibroblasts were very different between CPEO and MELAS patients although two other groups of researchers reported that Mn-SOD was over-expressed in MELAS patients [18,21]. While the mean enzyme activity of Mn-SOD in muscle fibroblasts of CPEO patients was increased 2- to 3-fold, we noted that the activity and protein levels of Mn-SOD in fibroblasts of MELAS patients were not changed but the activity and protein levels of catalase were lower than those of the normal controls (data not shown). These observations led us to suggest that distinct molecular and biochemical mechanisms are involved in the induction of Mn-SOD in muscle fibroblasts of CPEO patients but not in MELAS patients. Our findings that the mRNA, protein and activity levels of Mn-SOD are all increased in muscle fibroblasts of CPEO patients have provided direct evidence to support the notion that Mn-SOD gene induction is correlated with a defect in mitochondrial function. We suggest that the increased expression of Mn-SOD in muscle fibroblasts of CPEO patient may be an early response to respiratory chain dysfunction in genetically determined mitochondrial diseases and that such overexpression of the antioxidant gene may be effected through increased oxidative stress. Moreover, several nuclear genes have been identified to associate with dominant and recessive forms of CPEO characterized by accumulation of large-scale mtDNA deletions [6–8]. It has been suggested that the mtDNA replication/repair machinery is involved in the pathogenesis of autosomal dominant CPEO with mtDNA deletions. The functional roles of these nuclear gene mutations need to be further elucidated in the pathogenesis of CPEO syndrome. In conclusion, we demonstrated for the first time that the expression and enzyme activity levels of Mn-SOD, but not those of Cu,Zn-SOD, CAT and GPx, are increased in muscle fibroblasts of the patients with CPEO syndrome. The enzyme activity and mRNA ratios of Mn-SOD/CAT and Mn-SOD/GPx in muscle fibroblasts of the CPEO patients were significantly higher than those of the controls. Moreover, we showed that the intracellular levels of superoxide anions and hydrogen peroxide in muscle fibroblasts and 8-OHdG in muscle biopsies were significantly increased in patients with CPEO syndrome. On the basis of these findings, we suggest that an imbalance in antioxidant capacities between the free radical scavenging enzymes may be an
important etiological factor that contributes to the onset and progression of the mitochondrial disease.
Acknowledgments This work was supported by research grants from the National Science Council (NSC91-2320-B010-069), Executive Yuan and from the National Health Research Institutes (NHRI-EX91-9020BN), Taiwan, ROC.
References [1] E.A. Schon, E. Bonilla, S. DiMauro, Mitochondrial DNA mutations and pathogenesis, J. Bioenerg. Biomembr. 29 (1997) 131–149. [2] J.M. Shoffner, M.T. Lott, A.S. Voljavec, S.A. Soueidan, D.A. Costigan, D.C. Wallace, Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy, Proc. Natl. Acad. Sci. USA 86 (1989) 7952–7956. [3] M. Tanaka, M. Yoneda, K. Ohno, W. Sato, M. Yamamoto, I. Nonaka, S. Horai, T. Ozawa, Differently deleted mitochondrial genomes in maternally inherited chronic progressive external ophthalmoplegia, J. Inherit. Metab. Dis. 12 (1989) 359–362. [4] M. Zeviani, N. Bresolin, C. Gellera, A. Bordoni, M. Pannacci, P. Amati, M. Moggio, S. Servidei, G. Scarlato, S. DiDonato, Nucleus-driven multiple large-scale deletions of the human mitochondrial genome: a new autosomal dominant disease, Am. J. Hum. Genet. 47 (1990) 904–914. [5] Y.H. Wei, H.C. Lee, Mitochondrial DNA mutations and oxidative stress in mitochondrial diseases, Adv. Clin. Chem. 37 (2003) 83–128. [6] J. Kaukonen, J.K. Juselius, V. Tiranti, A. Kyttala, M. Zeviani, G.P. Comi, S. Keranen, L. Peltonen, A. Suomalainen, Role of adenine nucleotide translocator 1 in mtDNA maintenance, Science 289 (2000) 782–785. [7] J.N. Spelbrink, F.Y. Li, V. Tiranti, K. Nikali, Q.P. Yuan, M. Tariq, S. Wanrooij, N. Garrido, G. Comi, L. Morandi, L. Santoro, A. Toscano, G.M. Fabrizi, H. Somer, R. Croxen, D. Beeson, J. Poulton, A. Suomalainen, H.T. Jacobs, M. Zeviani, C. Larsson, Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria, Nat. Genet. 28 (2001) 223–231. [8] V. van Goethem, B. Dermaut, A. L€ ofgren, J.J. Jean-Jacques Martin, C.V. Broeckhoven, Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions, Nat. Genet. 28 (2001) 211–212. [9] G. Piccolo, P. Banfi, G. Azan, R. Rizzuto, R. Bisson, D. Sandona, G. Bellomo, Biological markers of oxidative stress in mitochondrial myopathies with progressive external ophthalmoplegia, J. Neurol. Sci. 105 (1991) 57–60. [10] Y.H. Wei, C.F. Lee, H.C. Lee, Y.S. Ma, C.W. Wang, C.Y. Lu, C.Y. Pang, Increase of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4977 bp-deleted mitochondrial DNA, Ann. N. Y. Acad. Sci. 928 (2001) 97–112. [11] C.Y. Pang, H.C. Lee, Y.H. Wei, Enhanced oxidative damage in human cells harboring A3243G mutation of mitochondrial DNA: implication of oxidative stress in the pathogenesis of mitochondrial diabetes, Diabet. Res. Clin. Pract. 54 (2001) S45–S56. [12] H. Carrier, F. Flocard, V. Tagliati, A.P. Arrigo, C. Godinot, Immunolabelling of mitochondrial superoxide dismutase and of
C.-Y. Lu et al. / Molecular Genetics and Metabolism 80 (2003) 321–329
[13]
[14]
[15]
[16]
[17]
Hsp60 in muscles harbouring a respiratory chain deficiency, Neuromuscul. Disord. 10 (2000) 144–149. S. DiGiovanni, M. Mirabella, M. Papacci, F. Odoardi, G. Silvestri, S. Servidei, Apoptosis and ROS detoxification enzymes correlate with cytochrome c oxidase deficiency in mitochondrial encephalomyopathies, Mol. Cell Neurosci. 17 (2001) 696–705. C.Y. Lu, H.C. Lee, H.J. Fahn, Y.H. Wei, Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin, Mutat. Res. 423 (1999) 11–21. D.R. Spitz, L.W. Oberley, An assay for superoxide dismutase activity in mammalian tissue homogenates, Anal. Biochem. 179 (1989) 8–18. V. Glisin, R. Crkvenjakov, C. Byus, Ribonucleic acid isolated by cesium chloride centrifugation, Biochemistry 13 (1974) 2633– 2637. M.K. Shigenaga, E.N. Aboujaoude, Q. Chen, B.N. Ames, Assays of oxidative DNA damage biomarkers 8-oxo-20 -deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by highperformance liquid chromatography with electrochemical detection, Methods Enzymol. 234 (1994) 16–33.
329
[18] H. Rusanen, K. Majamaa, I.E. Hassinen, Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A ! G mutation in mitochondrial DNA, Biochim. Biophys. Acta 1500 (2000) 10–16. [19] S. Pitk€anen, B.H. Robinson, mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase, J. Clin. Invest. 98 (1996) 345–351. [20] S. Raha, G.E. McEachern, A.T. Myint, B.H. Robinson, Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase, Free Radic. Biol. Med. 29 (2000) 170–180. [21] M. Filosto, P. Tonin, G. Vattemi, M. Spagnolo, N. Rizzuto, G. Tomelleri, Antioxidant agents have a different expression pattern in muscle fibers of patients with mitochondrial diseases, Acta Neuropathol. 103 (2002) 215–220. [22] T.C. Yen, K.L. King, H.C. Lee, S.H. Yeh, Y.H. Wei, Agedependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria, Free Radic. Biol. Med. 16 (1994) 207–214. [23] H.C. Lee, P.H. Yin, C.Y. Lu, C.W. Chi, Y.H. Wei, Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells, Biochem. J. 348 (2000) 425–432.
Increased expression of manganese-superoxide dismutase in fibroblasts of patients with CPEO syndrome Ching-You Lu,a Edward K. Wang,b Hsin-Chen Lee,c Huey-Jen Tsay,d and Yau-Huei Weia,* a
Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC b Section of Peripheral Neurology, Taipei Veterans General Hospital, Taipei 112, Taiwan, ROC c Department of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan, ROC d Institute of Neuroscience, National Yang-Ming University, Taipei 112, Taiwan, ROC Received 22 May 2003; received in revised form 31 July 2003; accepted 4 August 2003
Abstract Alterations in the expression of free radical scavenging enzymes and production of reactive oxygen species (ROS) in tissue cells may contribute to the pathogenesis of mitochondrial diseases such as chronic progressive external ophthalmoplegia (CPEO) syndrome. Since the mitochondria with impaired respiratory function in affected tissues generate more ROS via electron leakage, we examined the expression levels of free radical scavenging enzymes in primary culture of muscle fibroblasts of eight patients with CPEO syndrome. The results showed that the enzyme activity and protein levels of Mn-SOD of the fibroblasts from CPEO patients were significantly increased but those of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) were not increased compared with controls. A similar pattern was observed in the mRNA levels of Mn-SOD and GPx in muscle fibroblasts of all CPEO patients. The activity ratios of Mn-SOD/catalase and Mn-SOD/GPx in muscle fibroblasts of the CPEO patients were increased 1.7–3.4 and 1.8- to 5.3-fold, respectively, compared to those of the controls. Moreover, by using flow cytometry we found that the production of O 2 and H2 O2 in the fibroblasts was about 2 times higher than those of controls. The 8-OHdG/dG ratios in total DNA of muscle biopsies from three CPEO patients were much higher than those of age-matched controls as determined by high performance liquid chromatography (HPLC). In the light of these findings, we suggest that the increase in expression of Mn-SOD, ROS production and oxidative damage in affected tissues may play an important role in the pathogenesis and progression of the CPEO syndrome. Ó 2003 Elsevier Inc. All rights reserved. Keywords: CPEO syndrome; Mitochondrial DNA; Free radical scavenging enzyme; Mn-superoxide dismutase; Oxidative stress; Muscle fibroblasts
Introduction Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial encephalomyopathies, which is characterized by myopathy, progressive limitation of eye movement due to weakness of the extraocular muscle [1]. The disease can be either sporadic [2] or transmitted through maternal lineage [3] or of autosomal dominant inheritance [4]. Many of these symptoms are associated with ragged-red fibers in muscle, which are formed by the accumulation of structurally abnormal mitochondria with paracrystalline inclusions [1]. Two types of mutation in mitochondrial
* Corresponding author. Fax: +886-2-2826-4843. E-mail address: [email protected] (Y.-H. Wei).
1096-7192/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2003.08.005
DNA (mtDNA) have been found in muscle of CPEO patients. Most of the patients with CPEO harbor largescale deletions of mtDNA and a portion of the remaining CPEO patients harbor the A3243G mutation in the tRNALeuðUURÞ gene, which is more commonly associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) [1,5]. Recently, several nuclear genes have been identified to associate with dominant and recessive forms of CPEO characterized by accumulation of large-scale mtDNA deletions, including the genes coding for adenine nucleotide translocator 1 (ANT1) at locus 4q34–35 [6], a putative mitochondrial helicase (Twinkle) at locus 10q24 [7], and the mitochondrial DNA polymerase c at locus 15q22–26 [8]. Mitochondrial respiratory chain dysfunction in affected tissues of patients with mitochondrial diseases not
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only results in decreased energy production, but also causes increased generation of reactive oxygen species (ROS) with consequent oxidative stress and cellular damage [5]. Increased lipid peroxidation and reduced glutathione (GSH) level have been observed in blood plasma and erythrocytes of patients with CPEO syndrome [9]. Moreover, it has been demonstrated that the specific contents of 8-OHdG and lipid peroxides are significantly increased in cybrids harboring a high level of the 4977 bp-deleted mtDNA as compared with those of the control cybrids [10]. The ratio of glutathione disulfide/reduced glutathione (GSSG/GSH) and lipid peroxide content in the cybrids harboring >95% A3243G mutant mtDNA were significantly higher than those of control cybrids containing undetectable mutant mtDNA [11]. These findings have provided strong support to the notion that increased oxidative damage to vital biomolecules in affected tissues of CPEO patients is a result of the elevation of intracellular oxidative stress elicited by mitochondrial dysfunction in the target cells harboring mtDNA mutations [5]. To cope with the free radicals and ROS by-products generated from the mitochondrial respiratory chain, human cells have developed an antioxidant defense system consisting of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and an array of low-molecular-weight antioxidants including ascorbic acid, a-tocopherol, and GSH. Mn-SOD, located in the mitochondria, and Cu,Zn-SOD, located in the cytoplasm, catalyze the dismutation of superoxide anions into hydrogen peroxide and molecular oxygen. Then, GPx uses reduced glutathione to reduce hydrogen peroxide or CAT converts two molecules of hydrogen peroxide into water and oxygen. A fine balance among the free radical scavenging enzymes is necessary to prevent the oxidative damage to vital biomolecules in tissue cells. Recently, increased immuno-staining of MnSOD and Cu,Zn-SOD has been reported in cytochrome c oxidase (COX)-deficient muscle fibers in patients with
mtDNA mutations and respiratory chain defects [12]. Moreover, it was reported that the strong immunoreactivity to the Mn-SOD and Cu,Zn-SOD in deficient fibers correlated well with the presence of apoptotic nuclei [13]. These observations suggest that over-expression of Mn-SOD and Cu,Zn-SOD alone is not sufficient to dispose of increased ROS and may even induce or promote apoptotic cell death in fibers with defective respiratory function. We therefore hypothesized that alterations in the expression of free radical scavenging enzymes or increased intracellular level of ROS in affected tissues may contribute to the pathogenesis of mitochondrial disease such as CPEO syndrome. To test this hypothesis, we cultured muscle fibroblasts from eight patients with CPEO syndrome and examined the enzyme activities and mRNA levels of Mn-SOD, CAT, and GPx. The 8-OHdG content in the fibroblasts was also measured as an index of oxidative damage to DNA. We found an increase in Mn-SOD expression, ROS production, and oxidative DNA damage in muscle fibroblasts of these CPEO patients. The results obtained in this study have provided evidence to support the hypothesis that endogenous oxidative stress elicited by imbalanced expression of free radical scavenging enzymes in affected tissues of the patients may play a role in the pathogenesis and progression of CPEO syndrome.
Materials and methods Patients Muscle biopsies were procured from eight patients with CPEO syndrome and control subjects with their consent by one of the authors (E. K. Wang) with the assistance of attending physicians at Taipei Veterans General Hospital. The clinical features and laboratory findings of the CPEO patients are summarized in Table 1. The most common clinical symptoms of these
Table 1 Clinical features of the patients with CPEO syndrome examined in this study Clinical features
Patients 1
2
3
4
5
6
7
8
Age Sex Age of onset Ptosis External ophthalmoplegia Muscle biopsies ragged-red fibers MtDNA mutation in muscle fibroblasts Large-scale deletion Size (bp) A3243G point mutation Proportion of the mutated mtDNA (%) in fibroblasts
46 F 34 + + + +
46 F 26 + + + +
61 M 50 + + + +
55 F 45 + + + )
63 M 56 + + + )
50 F 30 + + + +
26 F 18 + + + )
26 F 15 + + + +
4977 ) 6.5
NA ) NA
4977 ) 4.5
+ 0.75
+ 0.75
4366 ) 20
+ 16
6190 ) 12
NA: Not available.
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patients were external ophthalmoplegia, ptosis, muscle weakness, and exercise intolerance. All of the 8 CPEO patients had ragged-red fibers in skeletal muscle upon staining with modified Gomori trichrome. Electron micrographs revealed large accumulations of abnormal mitochondria in the subsarcolemmal regions of the muscle fibers of these patients. Cell lines and culture condition Primary cultures of fibroblasts were established from muscle biopsies of patients with CPEO syndrome and healthy subjects of different ages. The muscle fibroblasts were grown in DulbeccoÕs modified EagleÕs medium (DMEM, Gibco-BRL, Bethesda, MD) with the supplement of 15% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 10 lg/ml pyruvate, and 5 lg/ml uridine. The cells were grown to confluence before use in a 150 cm2 petri dish in a growth chamber set at 37 °C under humidified atmosphere of 5% CO2 and 95% air. Assay of the activities of free radical scavenging enzymes To determine the enzyme activities of Cu,Zn-SOD, Mn-SOD, CAT, and GPx, muscle fibroblasts were trypsinized and washed with ice-cold phosphate-buffered saline (PBS, pH 7.3) and the cell pellets were collected by centrifugation. The cells were suspended in a pre-cooled NEP buffer (150 mM NaCl, 0.5 mM EDTA, and 100 mM phosphate buffer, pH 7.5) containing 0.25% sodium cholate and protease inhibitors (Boehringer Mannheim, Germany). The cell suspension was then mixed with an equal volume of glass beads and vortex-mixed vigorously for 8 intervals of 10 s per round, separated by 30 s cooling intervals. The cell lysate was spun down at 800g for 5 min, and the supernatant was collected for the assay of the activities of free radical scavenging enzymes. CAT activity was determined by monitoring the rate of decomposition of H2 O2 from the decrease in absorbance at 240 nm. GPx activity was determined using a coupled assay in which enzyme activity is proportional to the rate of NADPH oxidation as described by Lu et al. [14]. Total SOD activity was assayed by monitoring nitroblue tetrazolium (NBT) reduction according to Spitz and Oberley [15] with some modifications. The inhibition by SOD of NBT reduction by O 2 in the aerobic xanthine/xanthine oxidase system was followed by change of absorbance at 560 nm. One unit of SOD is defined as the amount of enzyme that causes 50% inhibition of NBT reduction under the assay condition described. Mn-SOD activity was differentiated from Cu,Zn-SOD by its resistance to NaCN. In the absence of NaCN, total SOD activity was measured and the MnSOD activity was assayed by monitoring NBT reduction in the presence of 5 mM NaCN.
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Determination of protein levels of free radical scavenging enzymes The cell lysate obtained from above-mentioned preparation was subjected to Western blot analysis of the protein levels of free radical scavenging enzymes. An aliquot of 25 lg of the cell lysate was subjected to 10% SDS–PAGE and transferred onto a Hybond nitrocellulose membrane (Amersham–Pharmacia Biotech, Little Chalfont, Buckinghamshire, England). After blocking for 1 h with 5% dried fat-free milk in Tris-buffered saline-Tween (TBST) buffer (20 mM Tris–HCl, pH 7.6, 0.9% NaCl, and 0.2% Tween 20), the membrane was incubated for 2 h with the primary antibody at room temperature. After washing three times with TBST, the blot was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then an enhanced chemiluminescence detection kit (Amersham–Pharmacia Biotech) was used. Autoradiograph of the protein signals in the Western blot was obtained by exposure of a Kodak X-ray film. The relative protein content of each free radical scavenging enzyme was normalized with that of GAPDH. Mouse anti-GAPDH and mouse anti-Mn-SOD monoclonal antibody were purchased from CHEMICON international (Temecula, CA), and sheep anti-human Cu, Zn-SOD antibody was bought from Biodesign International (Saco, Maine). RNA isolation and Northern hybridization Total RNA was isolated from muscle fibroblasts cultured under the above-mentioned condition [16]. Each RNA sample (30 lg) was dissolved in 1 ll of 10 MOPS running buffer, 3.5 ll of 12.3 M formaldehyde, and 10 ll deionized formamide and then denatured by incubation at 55 °C for 30 min. The RNA was electrophoresed on a 1.0% agarose denaturing gel and blotted overnight onto a nylon membrane in a 6 SSC buffer. The RNA blots were UV cross-linked and immersed completely in the 6 SSC buffer and prehybridized at 65 °C for 15 min in the rapid hybridization buffer. Purified cDNAs were labeled with [a-32 P]dCTP by random priming using a ready prime kit (Amersham Bioscience, Buckinghamshire, UK). Hybridization was performed at 65 °C for 2 h in the rapid hybridization buffer plus the cDNA probe of either Mn-SOD, CAT or GPx and washed with 2 SSC buffer containing 0.1% SDS at room temperature for 15 min. The blots were then washed twice with 0.1 SSC buffer/0.1% SDS at 65 °C for 15 min. Autoradiographs were obtained by exposure for 24–30 h of a Kodak X-ray film with an intensifying screen at )70 °C. The amount of the mRNA of each free radical scavenging enzyme was calculated on the basis of the intensity of its band in the RNA blot normalized by simultaneous hybridization with a mouse
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muscle b-actin cDNA probe. The cDNA probes of human Mn-SOD (546 bp), CAT (919 bp), and GPx (478 bp) were cloned by PCR using the published cDNA sequences of the respective genes, and the b-actin cDNA probe was purchased from Stratagene (La Jolla, CA). Flow cytometric measurement of superoxide anion and hydrogen peroxide The intracellular level of superoxide anions was measured by flow cytometry using hydroethidine (HE; Molecular Porbes, Eugene, OR). HE is a non-fluorescent compound that can diffuse through cell membranes, which then reacts rapidly with O 2 to form the red fluorescent product of ethidium. On the other hand, the intracellular level of H2 O2 was measured by use of the fluorescent dye dihydro-20 ,70 -dichlorofluorescin diacetate (H2 DCF-DA; Molecular Probes), which is converted to green fluorescent 20 ,70 -dichlorofluorescein (DCF) after reacting with H2 O2 . Usually, HE or DCFH-DA was added to the culture dish containing approximately 1 106 cells in DMEM (without phenol red). After 15 min of incubation at 37 °C, cells were kept on ice for immediate analysis on a FACScan flow cytometer (Becton–Dickinson, Bedford, MA) equipped with a 488 nm argon laser. The excitation wavelength was set at 488 nm. The observation wavelength of 530 nm was chosen for DCF green fluorescence and 585 nm for ethidium red fluorescence and the intensities of emitted fluorescence were collected on FL1 and FL2 channels, respectively. Data were acquired and analyzed using the Cell Quest software (Becton–Dickinson).
Analysis of 8-OHdG content in total DNA from muscle tissues The relative amount of 8-OHdG in total DNA of muscle biopsies from CPEO patients and control subjects was determined by the method developed by Shigenaga et al. [17] with modifications as described previously by Lu et al. [14].
Results Morphological characteristics of the muscle fibroblasts of CPEO patients The growth kinetics and morphological characteristics of the muscle fibroblasts of CPEO patients were quite different from those of the muscle fibroblasts of controls. It took 2 weeks for the muscle fibroblasts of controls to grow to confluence after subculture at 1:3 split. But at least 3 weeks were required for the muscle fibroblasts of our CPEO patients to grow to confluence after 1:3 split at subculture. Moreover, the muscle fibroblasts of all the CPEO patients examined showed significantly larger size and more flattened and extended morphology compared with the muscle fibroblasts of controls upon examination by a light microscope. The maximal number of muscle fibroblasts from CPEO patients was less than that of muscle fibroblasts of controls. On the other hand, muscle fibroblasts of the CPEO patients exhibited many more vacuoles and granular particles, which were clustered near the area surrounding the nucleus (Fig. 1).
Fig. 1. Morphology of muscle fibroblasts from CPEO patients and healthy subjects. A represents normal muscle fibroblasts. B–F show the muscle fibroblasts from patients CPEO2, CPEO3, CPEO4, CPEO5, and CPEO6 at a magnification of 150.
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MtDNA mutation in the muscle fibroblasts of CPEO patients Molecular analysis showed that mtDNA deletions of different sizes existed in muscle fibroblasts of patients CPEO1, CPEO2, CPEO3, CPEO6, and CPEO8 (Table 1). On the other hand, CPEO4, CPEO5, and CPEO7 patients harbored the A3243G point mutation in the mitochondrial tRNALeuðUURÞ gene. It should be noted that although the muscle biopsies from CPEO patients usually had high proportion of mutant mtDNA (above 60%), the proportion of mutant mtDNA was much lower in cultured muscle fibroblasts as compared to that of the corresponding muscle biopsies. A similar decline in the proportion of mtDNA with A3243G mutation was previously observed in cultured muscle fibroblasts compared with muscle biopsies [18]. Activities of free radical scavenging enzymes in muscle fibroblasts The enzyme activities of Cu,Zn-SOD, Mn-SOD, CAT, and GPx in muscle fibroblasts from the eight CPEO patients are summarized in Table 2. The activities of Mn-SOD in muscle fibroblasts of the CPEO patients were approximately 2-fold higher compared with those of controls. By contrast, the activities of Cu,Zn-SOD, CAT or GPx did not show much changes. Upon further analysis, we found that the activity ratios of Mn-SOD/ CAT and Mn-SOD/GPx in muscle fibroblasts from CPEO patients were significantly higher (p < 0:01) than the corresponding values of the controls (Figs. 2A and B). The ratios of enzyme activity between the mutant and control fibroblasts were 1.7–3.4 for Mn-SOD/CAT and 1.8–5.3 for Mn-SOD/GPx, respectively.
Fig. 2. The enzyme activity ratios of Mn-SOD/catalase (A) and MnSOD/103 GPx (B) of CPEO patients and controls. *Indicates significant difference compared with those of the control muscle fibroblasts, p < 0:01. The box-whisker plot made by using the Sigma Plot software extended from the 25th to the 75th percentile of interquartile range (IQR). The centerline indicates the median of enzyme activity ratio of Mn-SOD/catalase or Mn-SOD/GPx in CPEO patients and controls. The whiskers at the end of each box are defined as the 75th percentile of +1.5 IQR and the 25th percentile of –1.5 IQR. The black dots shown on the two plots represent the activity ratios outside the 1.5 IQR.
Protein levels of free radical scavenging enzymes in muscle fibroblasts The enzyme protein levels assessed by Western blotting were found to change in parallel with the change of the activities of free radical scavenging enzymes in the fibroblasts (Fig. 3). The Mn-SOD protein level in fibroblasts of the seven CPEO patients were 3- to 6-fold higher than those of the healthy subjects, but the protein
Fig. 3. Protein levels of different antioxidant enzymes from muscle fibroblasts of CPEO patients. GAPDH was used as internal standard in this Western blot analysis. Lanes 1 and 2 represent muscle fibroblasts from healthy subjects. Lanes 3–9 indicate muscle fibroblasts from patients CPEO1, CPEO2, CPEO4, CPEO5, CPEO6, CPEO7, and CPEO8, respectively.
Table 2 Comparison of the activities of free radical scavenging enzymes in muscle fibroblasts from the patients with CPEO syndrome with those of agematched controls Subject
Cu,Zn-SOD (unit/mg)
Mn-SOD (unit/mg)
Catalase (103 unit/g)
GPx (unit/g)
CPEO (n ¼ 8) Control (n ¼ 5)
149 68 118 24
260 24 116 34
6.2 1.3 6.6 1.6
56.0 15.0 65.9 6.7
The enzyme activities are expressed as means SD. * Significantly different from muscle fibroblasts of controls, p < 0:001, using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined were primary cultures between three and six passages.
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levels of Cu,Zn-SOD did not show any significant change. RNA levels of free radical scavenging enzymes in muscle fibroblasts To examine gene expression of Mn-SOD, CAT, and GPx in muscle fibroblasts from the CPEO patients and controls, we measured the mRNA levels of these antioxidant enzymes. The Northern blot analysis of the expression of these free radical scavenging enzymes is shown in Fig. 4. The mRNA levels of the enzymes were determined by densitometric scanning of the autoradiographs of Northern blots. The results are expressed in arbitrary units after normalization with the
intensity of the b-actin mRNA band. The mRNA levels of Mn-SOD, CAT, and GPx in muscle fibroblasts of the CPEO patients are summarized in Table 3. The changes in mRNA levels of Mn-SOD and GPx were similar to those observed for the activities and protein levels of the corresponding enzymes. In contrast to the activity, the mRNA level of CAT was also increased in muscle fibroblasts of CPEO patients. The mRNA levels of Mn-SOD and CAT in muscle fibro-
RNA ratio of Mn-SOD/CAT
326
4
A A
#
3
2
1
0
RNA ratio of Mn-SOD/GPx
2
B B
# 1
0
Control Fig. 4. The mRNA levels of Mn-SOD, catalase, and GPx in muscle fibroblasts of the patients with CPEO syndrome. Total RNA was extracted and analyzed by Northern blot and hybridized with 32 P-labeled cDNA probes of Mn-SOD, catalase, GPx, and b-actin as described in Materials and methods. The b-actin mRNA was used as an internal standard for normalizing the amount of RNA in each lane. The intensity of each band was expressed in arbitrary scale because it was determined by different duration of exposure of the autoradiographs and by cDNA probes of different size. Lanes 1–8 indicate the mRNA levels of muscle fibroblasts from the eight CPEO patients, and lanes 9 and 10 show the mRNA levels of Mn-SOD of the muscle fibroblasts from two healthy subjects, respectively.
CPEO
Fig. 5. The mRNA ratios of Mn-SOD/catalase (A) and Mn-SOD/GPx (B) in muscle fibroblasts of CPEO patients and controls. # Indicates significant difference compared with those of the control fibroblasts, p < 0:01. The box-whisker plots made by using the Sigma Plot software extended from the 25th to the 75th percentile of interquartile range (IQR). The centerline indicates the median of the activity ratio Mn-SOD/catalase or Mn-SOD/GPx in CPEO patients and controls. The whiskers at the end of each box are defined as the 75th percentile of +1.5 IQR and the 25th percentile of )1.5 IQR. The black dots shown on the two plots represent the activity ratios outside the 1.5 IQR.
Table 3 Comparison of the mRNA levels of Mn-SOD, catalase and glutathione peroxidase of muscle fibroblasts of patients with CPEO syndrome with those of age-matched controls Subject
Mn-SOD
Catalase
GPx
(Intensity ratio relative to that of b-actin) CPEO (n ¼ 8) Control (n ¼ 5)
0.68 0.17 0.22 0.06
0.34 0.07 0.16 0.04
0.69 0.20 0.72 0.09
Values for mRNAs of free radical scavengers Mn-SOD, catalase and GPx were normalized with the level of b-actin RNA. The data are presented as means SD. * Significantly (p < 0:001) compared with muscle fibroblasts of controls using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined were primary cultures between three and six passages.
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blasts of CPEO patients were approximately 3- and 2-fold, respectively, higher than those in the muscle fibroblasts of controls. We then examined the ratios of the mRNA level between Mn-SOD and CAT or between Mn-SOD and GPx. The ratios of mRNA level of Mn-SOD/CAT in the muscle fibroblasts of CPEO patients were higher than those of the controls but the difference did not reach statistical significance (Fig. 5A). The value was between 1.3 and 3.3 for CPEO patients compared with 1.38 in the controls. On the other hand, the mRNA ratio of Mn-SOD/GPx in the muscle fibroblasts of CPEO patients was increased 2- to 4-fold as compared with those of the controls (Fig. 5B). Intracellular levels of superoxide anions and hydrogen peroxide The contents of superoxide anions and hydrogen peroxide in muscle fibroblasts from the CPEO patients are listed in Table 4. We demonstrated that the muscle fibroblasts of CPEO patients exhibited a higher intracellular level of superoxide anions and hydrogen peroxide compared with muscle fibroblasts of controls.
Table 4 The intracellular levels of superoxide anions and hydrogen peroxide in muscle fibroblasts of patients with CPEO syndrome Subject
Superoxide anions (Ethidium intensity)
Hydrogen peroxide (DCF intensity)
CPEO (n ¼ 8) Control (n ¼ 5)
186 60
235 82
116 21
126 21
The intracellular contents of superoxide anions and hydrogen peroxide in muscle fibroblasts of patients with CPEO syndrome were measured by staining with hydroethidine and dihydro-20 ,70 -dichlorofluorescin diacetate and then analyzing by flow cytometry. The contents of superoxide anions and hydrogen peroxide are presented as means SD. * Significantly different from muscle fibroblasts of controls, p < 0:01, using the StudentÕs t-test. The ages of controls were between 25 and 70 years old. The muscle fibroblasts examined in this study were primary cultures between three and six passages.
Table 5 Comparison of the 8-OHdG content in total cellular DNA of muscle biopsies of CPEO patients and controls Subject
8-OHdG/106 dG ratio
CPEO (n ¼ 3) Control (n ¼ 7)
50.4 23.8 5.8 1.6
* Significantly different from the controls, p < 0:05, using the StudentÕs t-test. Control data were obtained from 7 subjects receiving surgical operations who had none of the known mitochondrial diseases with ages between 40 and 70 years old.
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The 8-OHdG content in skeletal muscle of CPEO patients The oxidative damage to DNA in muscle biopsies of CPEO patients was found to be more severe than that of controls as indicated by the significant increase in the level of 8-OHdG/dG. As shown in Table 5, the levels of 8-OHdG/dG in muscle biopsies of CPEO patients were significantly higher than those of the controls.
Discussion In this study, we demonstrated for the first time that Mn-SOD was up-regulated at RNA, protein, and activity levels in muscle fibroblasts from CPEO patients. The activity and protein levels of Mn-SOD, but not those of Cu,Zn-SOD, CAT, and GPx, in muscle fibroblasts of the patients with CPEO syndrome were significantly higher than those of the controls. Moreover, Mn-SOD mRNA levels in muscle fibroblasts of CPEO patients were also significantly higher than those of the controls. The observation of a concurrent increase in gene expression and enzyme activity of Mn-SOD in muscle fibroblasts of CPEO patients is in agreement with previous reports on other mitochondrial disorders [19–22]. It has been reported that the severity of Complex I enzyme deficiency correlated with the increase of production of O 2 and the induction of gene expression of Mn-SOD [19]. In addition, it has been reported that the expression of MnSOD, but not of Cu,Zn-SOD, is increased in the skeletal muscle with ragged-red fibers in a number of patients with mitochondrial encephalomyopathies [20,21]. What causes the induction of Mn-SOD and whether there is a correlation with the severity of the disease have remained unclear. The elevation in gene expression and activity of Mn-SOD in muscle fibroblasts of the CPEO patients may be one of the responses of the cells to cope with overproduction of free radicals. Recently, Filosto et al. [21] conducted an immunohistochemical study on muscle biopsies from patients with mitochondrial diseases. They showed that the induction of gene expression of antioxidant enzymes correlates with the extent of mitochondrial proliferation. In addition, we have recently demonstrated that enhanced oxidative stress can lead to an increase in mitochondrial mass and mtDNA content of human lung fibroblasts [23]. These observations suggest a possible mechanism by which the elevated ROS production from defective electron transport chain results in enhanced oxidative stress, which subsequently causes mitochondrial proliferation and overexpression of the mitochondrial Mn-SOD. Indeed, we found that muscle fibroblasts from CPEO patients had about 1.6- and 1.9-fold higher levels of O 2 and H2 O2 as reported by HE and H2 DCF-DA, respectively. In addition, the oxidative damage to DNA, as indicated by the 8-OHdG/106 dG level, in muscle of CPEO patients
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was much more extensive than that in muscle of controls or patients with other types of diseases that are not associated with mtDNA mutation. Patients with MELAS or CPEO syndromes harboring A3243G mutation of mtDNA manifest very different clinical features. Thus, MELAS patients rarely present with ptosis and patients with CPEO syndrome never exhibited stroke-like episodes. We found that the activities and protein levels of free radical scavenging enzymes in muscle fibroblasts were very different between CPEO and MELAS patients although two other groups of researchers reported that Mn-SOD was over-expressed in MELAS patients [18,21]. While the mean enzyme activity of Mn-SOD in muscle fibroblasts of CPEO patients was increased 2- to 3-fold, we noted that the activity and protein levels of Mn-SOD in fibroblasts of MELAS patients were not changed but the activity and protein levels of catalase were lower than those of the normal controls (data not shown). These observations led us to suggest that distinct molecular and biochemical mechanisms are involved in the induction of Mn-SOD in muscle fibroblasts of CPEO patients but not in MELAS patients. Our findings that the mRNA, protein and activity levels of Mn-SOD are all increased in muscle fibroblasts of CPEO patients have provided direct evidence to support the notion that Mn-SOD gene induction is correlated with a defect in mitochondrial function. We suggest that the increased expression of Mn-SOD in muscle fibroblasts of CPEO patient may be an early response to respiratory chain dysfunction in genetically determined mitochondrial diseases and that such overexpression of the antioxidant gene may be effected through increased oxidative stress. Moreover, several nuclear genes have been identified to associate with dominant and recessive forms of CPEO characterized by accumulation of large-scale mtDNA deletions [6–8]. It has been suggested that the mtDNA replication/repair machinery is involved in the pathogenesis of autosomal dominant CPEO with mtDNA deletions. The functional roles of these nuclear gene mutations need to be further elucidated in the pathogenesis of CPEO syndrome. In conclusion, we demonstrated for the first time that the expression and enzyme activity levels of Mn-SOD, but not those of Cu,Zn-SOD, CAT and GPx, are increased in muscle fibroblasts of the patients with CPEO syndrome. The enzyme activity and mRNA ratios of Mn-SOD/CAT and Mn-SOD/GPx in muscle fibroblasts of the CPEO patients were significantly higher than those of the controls. Moreover, we showed that the intracellular levels of superoxide anions and hydrogen peroxide in muscle fibroblasts and 8-OHdG in muscle biopsies were significantly increased in patients with CPEO syndrome. On the basis of these findings, we suggest that an imbalance in antioxidant capacities between the free radical scavenging enzymes may be an
important etiological factor that contributes to the onset and progression of the mitochondrial disease.
Acknowledgments This work was supported by research grants from the National Science Council (NSC91-2320-B010-069), Executive Yuan and from the National Health Research Institutes (NHRI-EX91-9020BN), Taiwan, ROC.
References [1] E.A. Schon, E. Bonilla, S. DiMauro, Mitochondrial DNA mutations and pathogenesis, J. Bioenerg. Biomembr. 29 (1997) 131–149. [2] J.M. Shoffner, M.T. Lott, A.S. Voljavec, S.A. Soueidan, D.A. Costigan, D.C. Wallace, Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy, Proc. Natl. Acad. Sci. USA 86 (1989) 7952–7956. [3] M. Tanaka, M. Yoneda, K. Ohno, W. Sato, M. Yamamoto, I. Nonaka, S. Horai, T. Ozawa, Differently deleted mitochondrial genomes in maternally inherited chronic progressive external ophthalmoplegia, J. Inherit. Metab. Dis. 12 (1989) 359–362. [4] M. Zeviani, N. Bresolin, C. Gellera, A. Bordoni, M. Pannacci, P. Amati, M. Moggio, S. Servidei, G. Scarlato, S. DiDonato, Nucleus-driven multiple large-scale deletions of the human mitochondrial genome: a new autosomal dominant disease, Am. J. Hum. Genet. 47 (1990) 904–914. [5] Y.H. Wei, H.C. Lee, Mitochondrial DNA mutations and oxidative stress in mitochondrial diseases, Adv. Clin. Chem. 37 (2003) 83–128. [6] J. Kaukonen, J.K. Juselius, V. Tiranti, A. Kyttala, M. Zeviani, G.P. Comi, S. Keranen, L. Peltonen, A. Suomalainen, Role of adenine nucleotide translocator 1 in mtDNA maintenance, Science 289 (2000) 782–785. [7] J.N. Spelbrink, F.Y. Li, V. Tiranti, K. Nikali, Q.P. Yuan, M. Tariq, S. Wanrooij, N. Garrido, G. Comi, L. Morandi, L. Santoro, A. Toscano, G.M. Fabrizi, H. Somer, R. Croxen, D. Beeson, J. Poulton, A. Suomalainen, H.T. Jacobs, M. Zeviani, C. Larsson, Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria, Nat. Genet. 28 (2001) 223–231. [8] V. van Goethem, B. Dermaut, A. L€ ofgren, J.J. Jean-Jacques Martin, C.V. Broeckhoven, Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions, Nat. Genet. 28 (2001) 211–212. [9] G. Piccolo, P. Banfi, G. Azan, R. Rizzuto, R. Bisson, D. Sandona, G. Bellomo, Biological markers of oxidative stress in mitochondrial myopathies with progressive external ophthalmoplegia, J. Neurol. Sci. 105 (1991) 57–60. [10] Y.H. Wei, C.F. Lee, H.C. Lee, Y.S. Ma, C.W. Wang, C.Y. Lu, C.Y. Pang, Increase of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4977 bp-deleted mitochondrial DNA, Ann. N. Y. Acad. Sci. 928 (2001) 97–112. [11] C.Y. Pang, H.C. Lee, Y.H. Wei, Enhanced oxidative damage in human cells harboring A3243G mutation of mitochondrial DNA: implication of oxidative stress in the pathogenesis of mitochondrial diabetes, Diabet. Res. Clin. Pract. 54 (2001) S45–S56. [12] H. Carrier, F. Flocard, V. Tagliati, A.P. Arrigo, C. Godinot, Immunolabelling of mitochondrial superoxide dismutase and of
C.-Y. Lu et al. / Molecular Genetics and Metabolism 80 (2003) 321–329
[13]
[14]
[15]
[16]
[17]
Hsp60 in muscles harbouring a respiratory chain deficiency, Neuromuscul. Disord. 10 (2000) 144–149. S. DiGiovanni, M. Mirabella, M. Papacci, F. Odoardi, G. Silvestri, S. Servidei, Apoptosis and ROS detoxification enzymes correlate with cytochrome c oxidase deficiency in mitochondrial encephalomyopathies, Mol. Cell Neurosci. 17 (2001) 696–705. C.Y. Lu, H.C. Lee, H.J. Fahn, Y.H. Wei, Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin, Mutat. Res. 423 (1999) 11–21. D.R. Spitz, L.W. Oberley, An assay for superoxide dismutase activity in mammalian tissue homogenates, Anal. Biochem. 179 (1989) 8–18. V. Glisin, R. Crkvenjakov, C. Byus, Ribonucleic acid isolated by cesium chloride centrifugation, Biochemistry 13 (1974) 2633– 2637. M.K. Shigenaga, E.N. Aboujaoude, Q. Chen, B.N. Ames, Assays of oxidative DNA damage biomarkers 8-oxo-20 -deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by highperformance liquid chromatography with electrochemical detection, Methods Enzymol. 234 (1994) 16–33.
329
[18] H. Rusanen, K. Majamaa, I.E. Hassinen, Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A ! G mutation in mitochondrial DNA, Biochim. Biophys. Acta 1500 (2000) 10–16. [19] S. Pitk€anen, B.H. Robinson, mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase, J. Clin. Invest. 98 (1996) 345–351. [20] S. Raha, G.E. McEachern, A.T. Myint, B.H. Robinson, Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase, Free Radic. Biol. Med. 29 (2000) 170–180. [21] M. Filosto, P. Tonin, G. Vattemi, M. Spagnolo, N. Rizzuto, G. Tomelleri, Antioxidant agents have a different expression pattern in muscle fibers of patients with mitochondrial diseases, Acta Neuropathol. 103 (2002) 215–220. [22] T.C. Yen, K.L. King, H.C. Lee, S.H. Yeh, Y.H. Wei, Agedependent increase of mitochondrial DNA deletions together with lipid peroxides and superoxide dismutase in human liver mitochondria, Free Radic. Biol. Med. 16 (1994) 207–214. [23] H.C. Lee, P.H. Yin, C.Y. Lu, C.W. Chi, Y.H. Wei, Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells, Biochem. J. 348 (2000) 425–432.