Mutagenesis vol. 18 no. 6 pp. 497-503,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Mitochondrial impairment is accompanied by impaired oxidative DNA repair in the nucleus
1Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 212312, USA, 2Department of Life Sciences and Chemistry, Roskilde University, 4000 Roskilde, Denmark, 3Free Radical and Radiation Biology Graduate Program, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242, USA and 4Department of Cancer Genetics, Roswell Park Cancer Institute, Cell and Virus Building, Room 247, Elm and Carlton Streets, Buffalo, NY 14263, USA
5Present address: Department of Radiation Oncology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Depletion of the mitochondrial genome is involved in several human diseases, as well as in mitochondrial diseases induced by drug therapies used in the treatment of cancer and human immunodeficiency virus. In order to identify the molecular changes underlying the pathogenesis of mitochondrial diseases, we determined the oxidative status of a human cell line following depletion of the mitochondrial genome (denoted
0 cells). Our analysis revealed that 0 cells contained 
10-fold lower levels of superoxide than parental cells (+), as detected by oxidation of dihydroethidium. No concurrent decrease in oxidation of hydrogen peroxide, detected using the dye dichloroflorescein diacetate, was observed in 0 cells. Depletion of the mitochondrial genome did not affect either the expression of superoxide dismutase or its activity. However, catalase expression and its activity decreased in 0 cells. In addition, glutathione peroxidase activity was higher in 0 cells compared with +. 0 cells showed increased lipid peroxidation, increased oxidative damage to the nuclear genome and impaired DNA repair. Our data illustrate the importance of the mitochondrial genome and its function to the cellular oxidative environment and nuclear genome instability. It also provides insights into the development of mitochondrial disease as a consequence of cancer therapy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mitochondrial toxicity is an adverse consequence of an array of drugs currently used in the treatment of diseases. Nucleoside reverse transcriptase inhibitors (NRTIs), highly effective antiretroviral drugs used for human immunodeficiency virus (HIV) therapy, are one class of drugs that cause mitochondrial toxicity (Jain et al., 2001
; Lewis et al., 2001; Rowe et al., 2001; White, 2001). These antiretroviral drugs include zidovudine, zalcitabine and fialuridine (Rowe et al., 2001). Studies suggest that NRTI treatment of HIV patients causes mitochondrial myopathy, including ragged red fibers, cardiac myopathy with cardiac dilation and failure, neuropathy and lactic acidosis (Lewis et al., 2001; Rowe et al., 2001). The molecular mechanism(s) underlying these syndromes is not yet known. However, recent studies demonstrate that NRTI lead to depletion of the mitochondrial genome. This is because NRTI can either directly inhibit mitochondrial (mt)DNA polymerase 
, which is involved in mtDNA replication, or be misincorporated into mtDNA causing termination of mtDNA synthesis (Jain et al., 2001; Lewis et al., 2001; Rowe et al., 2001; White, 2001). A number of chemotherapeutic drugs used for the treatment of cancer also inhibit mtDNA replication and cause depletion of the mitochondrial genome, including intercalating agents, non-intercalating DNA-binding drugs, DNA topoisomerase inhibitors and lipophilic cationic drugs (reviewed in Rowe et al., 2001). Our previous study demonstrated that the routinely used anticancer drug adriamycin also causes depletion of mtDNA (Singh et al., 1999). We have also demonstrated that loss of mtDNA did not alter apoptotic potential, although the cells lacking mtDNA became resistant to adriamycin and other chemotherapeutic agents (Singh et al., 1999).
The human mitochondrial genome is a 16.6 kb double-stranded circular DNA sequence that encodes 13 proteins that function in oxidative phosphorylation and ATP synthesis, as well as rRNAs and tRNAs (Anderson et al., 1981). The mitochondrial genome is exposed to significant oxidative stress, due to its proximity to participants in oxidative phosphorylation (Mathupala et al., 1997). In the process of oxidative phosphorylation, reactive oxygen species (ROS) are formed from molecular oxygen, which can damage proteins, lipids and DNA (Ames et al., 1993; Shigenaga et al., 1994; Breen and Murphy, 1995; Kang et al., 1998). These ROS intermediates include the oxidant superoxide and hydrogen peroxide, which is produced from the reduction of superoxide by superoxide dismutase (reviewed in Kang et al., 1998). Under normal physiological conditions, cells are protected from damage by these oxidative intermediates by antioxidant mechanisms (Kang et al., 1998; Yakes and Van Houten, 1997). However, under pathological conditions such as hypoxia or ischemia, increased levels of ROS are generated, which can result in extensive damage that may exceed the capacity of the cell repair systems (Yakes and Van Houten, 1997). In addition, mtDNA contains few non-coding regions and is not associated with the histone proteins that contribute to the protection of the nuclear genome (LeDoux et al., 1999). Oxidative damage to mtDNA has also been demonstrated to be more extensive than oxidative damage to nuclear DNA (Yakes and Van Houten, 1997). Recently, mutations in mtDNA have been shown to occur frequently in a variety of human cancers, including colorectal (Polyak et al., 1998), bladder (Fliss et al., 2002), head and neck (Fliss et al., 2002), lung (Fliss et al., 2002), liver (Nishikawa et al., 2000) and pancreatic (Jones et al., 2001) tumors. ROS have been proposed to function in the mtDNA mutations observed in these cancers (LeDoux et al., 1999; Fliss et al., 2002), as well as in the mitochondrial toxicity of therapeutic drugs (Jain et al., 2001).
To understand the basis of ‘acquired’ mitochondrial diseases caused by cancer therapeutic drugs, we examined the oxidative status of cells lacking the mitochondrial genome. Experiments described in this article suggest that mtDNA depletion results in a decreased level of superoxide radical. However, no change in hydrogen peroxide level was observed. Interestingly, we found that mtDNA depletion caused increased lipid peroxidation and oxidative damage to DNA. Our study implicates mitochondria-derived oxidative stress as involved in the pathology of mitochondrial diseases acquired due to cancer therapy.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell culture and reagents
Parental HeLa cells and a
0 variant that had been depleted of mtDNA by culture in ethidium bromide as described (Morais et al., 1994) were a generous gift from Dr Rejean Morais. The HeLa 0 variant was routinely tested for 0 status by southern and western blotting using DNA probes and antibodies raised against cytochrome c oxidase subunits I and II encoded by the mitochondrial genome. Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies Inc.) containing 10% heat-inactivated fetal bovine serum (Life Technologies Inc.), L-glutamine (Mediatech Inc., Herndon, VA), penicillin/streptomycin (PS) (50 IU/ml and 50 µg/ml, respectively) (Mediatech Inc.), sodium pyruvate (2 mM) and uridine (4 µg/ml) at 37°C in 10% CO2/90% air. Chemical reagents were obtained from Sigma-Aldrich Corp. (St Louis, MO) unless otherwise indicated.
Measurement of reactive oxygen species
Cells collected by trypsin treatment were washed with Dulbecco’s phosphate-buffered saline (DPBS) and resuspended in Hanks’ balanced salt solution (HBSS) without calcium, magnesium or phenol red but containing 0.5% fetal bovine serum and either menadione (0.1 or 1 mM), H2O2 (0.1 or 1 mM) or an equal volume of deionized water as a control. Cells were incubated at 37°C in 10% CO2/90% air for 1 h and washed with HBSS. Dichlorofluorescein diacetate (CM-H2DCFDA) and dihydroethidium (DHE) (Molecular Probes) were dissolved in dimethylsulfoxide (DMSO) to 1 mM. Control and H2O2-treated cells were resuspended in HBSS without calcium, magnesium or phenol red but containing 0.5% fetal bovine serum and CM-H2DCFDA at 10 µM. Control and menadione-treated cells were resuspended in HBSS without calcium, magnesium or phenol red but containing 0.5% fetal bovine serum and DHE at 10 µM. Control cells treated with DMSO (1%) alone were included. Cells were incubated with the dyes for 30 min and analyzed by flow cytometry as described previously (Hockenbery et al., 1993; Ha et al., 1997) using a Becton-Dickinson LSR. A previous study had demonstrated that the uptake and efflux of a similar fluorescent compound was not altered in 0 cells (Singh et al., 1999). 10 000 events were recorded for each sample.
Measurement of antioxidant activity
Catalase activity was measured by the spectrophotometric method of Beers and Sizer (Beers and Sizer, 1952; Worthington, 1993). Subconfluent cultures were collected by trypsin treatment, washed with phosphate-buffered saline (PBS) and cells were resuspended in 0.05 M potassium phosphate, pH 7.0, and subjected to three freeze/thaw cycles. Triton X-100 was added to a final concentration of 1%, lysates were mixed with one half volume of glass beads and vortexed three times for 10 s, with 1 min cooling on ice between cycles. Lysates were centrifuged for 5 min at 4°C at 16 000 g and protein concentration of supernatants was estimated using Bio-Rad Protein Assay Reagent compared with bovine serum albumin (BSA) standards. Equal volumes of cleared lysate were added to 0.06% H2O2 in 0.05 M potassium phosphate, pH 7.0, and the change in absorbance at 240 nm was recorded over 3 min. Superoxide dismutase activity was measured as described by Spitz and Oberley (1989). Cells were lyzed in a buffer containing 0.1 M potassium phosphate, 150 mM NaCl, 0.25% cholic acid, 0.57 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin and 1 µg/ml leupeptin and vortexed with glass beads as described by Lu et al. (1999). Western blot analysis was carried by using equal amounts of protein as described by Garfin (1990). Glutathione peroxidase (GPx) enzyme activity was measured following a previously published method (Lawrence and Burk, 1976).
Measurement of thiobarbituric acid reactivity
Thiobarbituric acid reactivity was measured as described by Bowie et al. (1997). Subconfluent cultures were collected by trypsin treatment, washed with ice-cold PBS and lyzed by three freeze/thaw cycles in sterile deionized water. Protein concentration of samples was estimated using Bio-Rad Protein Assay Reagent compared with BSA standards. 1',1',3',3'-Tetramethoxypropane (TMP) was diluted to 10 mM in 20 mM Tris–HCl, pH 7.5, and serially diluted for standards. Equal volumes (200 µl) of lysate or TMP standards were mixed with 800 µl of solution containing 4 mg/ml 2-thiobarbituric acid (TBA), 0.5% SDS and 9.4% glacial acetic acid and heated at 95°C for 1 h. Samples were cooled to room temperature, centrifuged for 10 min at 16 000 g and the absorbance of the supernatants was read at 532 nm. Concentrations of TBA-reactive substances were estimated by comparing values with a TMP standard curve. This method has been extensively used to measure lipid peroxidation (see Ochoa et al., 2003; Patockova et al., 2003; Sheu et al., 2003).
Analysis of DNA damage and repair using the Comet assay
The Comet assay was carried out essentially as described by McNamee et al. (1995). Four Lab-Tek II 2-well chambers (Life Technologies, Bethesda, MD) were attached to one Gel Bond film (BMA BioWhittaker Molecular Applications, Rockland, ME) with 1% liquefied normal melting point agarose. All subsequent steps were performed in subdued light. Upon gel solidification, cell suspensions were immersed in 0.75% low melting point agarose (39°C) and cast in the Lab-Tek II 2-well chambers. The gels were allowed to solidify for 15 min at 4°C. Following solidification cells were lyzed in ice-cold lysis buffer, without light, for 60 min at 4°C. Subsequent to lysis, the lysis buffer was decanted and gels were washed three times with Milli-Q water. Gels were then immersed in alkaline electrophoresis buffer and kept without light at 4°C for 40 min. Cells were electrophoresed at constant voltage (27 V) for 20 min at 4°C. Following electrophoresis, gels were washed twice in neutralization buffer (2 x 5 min), washed once with Milli-Q water and then placed in 70% ethanol for 90 min without light, before being left to dry overnight without light. Gels were stained with SYBR Gold Nucleic Acid Gel Stain. The gels were incubated with shaking and without light for 19 min. Subsequent to staining, the gels were rinsed with Milli-Q water and left to air dry overnight. Comets were visualized in a fluorescence microscope (Leica DM IRB, Filter cube N.2.1, code no. 513832, excitation filter BP 515–560). For cells treated with H2O2, 100 comets on each slide were scored based on tail length. Comets were given points according to four categories (0, no visible comet; 1, visible comet but clear illuminating core; 2, clear visible comet and no clear illuminating core; 3, comet appears as cloud and no core is visible) and the points used to produce an arbitrary DNA damage value ranging from 0 (no damage) to a total score of 300 (all damaged). Comet length was quantitated as the distance from the core to the tail of the comet.
H2O2 exposure
Approximately 2.5 x 104 cells were plated in 1 ml multi-well dishes (NUNC) 1 day prior to experiment. Cells were incubated overnight at 37°C and 5% CO2 to ensure proper plating. The medium was removed and cells were washed twice with DPBS. Cells were incubated at 4°C for 20 min in DMEM with Glutamax-1 (Gibco BRL Life Technologies) containing 100 µM H2O2 or in medium alone as a control. Following incubation, DMEM with Glutamax-1, 10% FBS, 1% PS, sodium pyruvate (2 mM) and uridine (4 µg/ml) was added to the cell samples. Samples were either incubated for 2 h at 37°C and 5% CO2 or immediately processed for Comet assay. For Comet assay assessment, the medium from each sample was removed and stored in 5 ml Falcon tubes. Cells were washed with water and subsequently washed twice in DPBS and recovered by trypsin treatment and incubation for 4–5 min at 37°C and 5% CO2. Cells were pooled with the medium in 5 ml Falcon tubes and centrifuged at 1000 g for 5 min. After centrifugation, the supernatant was removed and cells were resuspended in DMEM with Glutamax-1, 10% FBS and 1% PS. A small amount of each sample was used to perform the Comet assay.
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exposure
Cells were washed with DPBS and recovered by trypsin treatment and incubation for 2 min at 37°C and 5% CO2. Afterwards, cells were resuspended in DMEM with Glutamax-1, 10% FBS and 1% PS and counted in a counting chamber. Each sample contained approximately 3.6 x 105 0 cells and 7.6 x 105 + cells. Cell suspensions were centrifuged in Eppendorf tubes at 1000 g for 5 min and washed with DPBS. Cells were incubated at 37°C and 5% CO2 for 60 min in DMEM with Glutamax-1 containing 10 µM MNNG or in DMEM with Glutamax-1 alone as a control. Following incubation, cells were resuspended in DMEM with Glutamax-1, 10% FBS, 1% PS, sodium pyruvate (2 mM) and uridine (4 µg/ml) and a small amount was used to perform the Comet assay directly. The remaining volume of each sample was split into three wells in multi-well dishes and extra medium was added. The multi-well dishes were incubated for 24, 48 and 72 h, respectively. After approximately 24, 48 and 72 h incubation the medium was removed, cells were washed with DPBS and recovered as described previously. Cells were resuspended in DMEM with Glutamax-1, 10% FBS, 1% PS, sodium pyruvate (2 mM) and uridine (4 µg/ml) and a small amount of each sample was used to perform the Comet assay.
Statistical analysis
Results were subjected to Student’s t-test or Welch’s alternate t-test using the InStat biostatistics software package (GraphPad Inc.). Histograms from flow cytometry experiments were compared by the Kolmogorov–Smirnov test using the CellQuest (Becton-Dickinson) analysis software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mitochondrial dysfunction results in a decrease level of pro-oxidant
Mitochondria are the major cellular site of ROS production (reviewed in Ames et al., 1993
). Therefore, it has frequently been speculated that mitochondrial dysfunction may lead to increased ROS production (Wallace, 2001; Wei et al., 2001). We examined this possibility using the oxidation-sensitive dyes DHE and CM-H2DCFDA. Oxidation of these compounds, predominantly by superoxide or hydrogen peroxide, respectively, results in a change in their fluorescence that can be measured intracellularly by flow cytometry (Vanden Hoek et al., 1997). Ethidium fluorescence, which is generated by the oxidation of DHE, was decreased 10-fold in the 0 cells (Figure 1A, dotted line) relative to the parental + cells (Figure 1A, solid line), indicating lower levels of the pro-oxidant (superoxide radical) in cells with dysfunctional mitochondria. Treatment of 0 cells with menadione, which generates superoxide radicals (McCord and Fridovich, 1970), increased pro-oxidant production, as indicated by a shift to the right of the ethidium fluorescence intensity peak generated by the oxidation of DHE (data not shown). However, even in cells stimulated to generate superoxide, the fluorescence intensity of DHE did not reach the levels seen in + cells, either in the presence or absence of menadione (data not shown). In contrast, the fluorescence intensity of CM-H2DCFDA did not differ between 0 cells (Figure 1B, dotted line) and + cells (Figure 1B, solid line). We conclude that mitochondrial dysfunction alters the oxidative environment of cells and that depletion of mtDNA leads to a decrease in pro-oxidant status.
|
Catalase activity is decreased in cells with mitochondrial dysfunction
The differences in levels of ROS observed between
+ and 0 cells could be due to either alterations in the production of these intermediates or to changes in their catabolism. Therefore, we examined the latter possibility by analyzing the activity of the antioxidant enzymes SOD, catalase and GPx. SOD catalyzes the breakdown of superoxide to hydrogen peroxide, while catalase and GPX convert hydrogen peroxide to water (reviewed in Halliwell, 1978). No apparent difference in MnSOD or CuZnSOD protein level was observed (Figure 2B). Consistent with the expression level, no significant difference in SOD activity was noted between the + and 0 cellular extracts (Figure 2C). In contrast, catalase expression and activity were decreased in 0 cells relative to those in + cells (Figure 2A and B). In addition, GPx activity was increased in 0 cells (Figure 2C). Thus, we conclude that 0 cells have decreased catalase and increased GPx activity, while no significant change in SOD activity was observed in these cells.
|
Increased DNA damage due to mitochondrial impairment
The repair efficiency of the parental HeLa cells and the
0 derivative was evaluated using the Comet assay. No difference was observed between the untreated parental and 0 cells (Figure 3A). However, when cells were treated with hydrogen peroxide, 0 exhibited increased damage as compared with parental cells (Figure 3B). We also tested the sensitivity of 0 cells to an alkylating agent, MNNG. Both parental and 0 cells were equally sensitive to damage by the alkylating agent (Figure 3C and D). We conclude that mitochondrial impairment in 0 cells contributes to impaired ability to repair oxidative DNA damage in the nuclear genome induced by exposure to hydrogen peroxide.
|
Lipid peroxidation is increased in cells with mitochondrial dysfunction
Malondialdehyde, a product of the catabolism of peroxidized lipids, forms a pigmented reaction product with TBA that can be measured spectrophotometrically (Janero, 1990
). Levels of TBA-reactive species were measured in extracts of HeLa + and 0 cells to determine if mitochondrial dysfunction resulting from the depletion of mtDNA alters lipid peroxidation. TBA-reactive species were increased in 0 cells (Figure 4, solid bar) by 43% as compared with the + parental cells (Figure 4, open bar). We conclude that an increase in lipid peroxidation is associated with the impairment of mitochondrial function.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Recent studies have demonstrated that NRTIs and other chemotherapeutic agents are involved in the development of mitochondrial diseases due to depletion of the mitochondrial genome (Jain et al., 2001
; Lewis et al., 2001; Rowe et al., 2001). Depletion of mtDNA is also involved in a number of pediatric syndromes (Wallace et al., 1995). In addition to depletion, a large number of mutations have been observed in the mitochondrial genome of many cancers examined (Polyak et al., 1998; Nishikawa et al., 2000; Fliss et al., 2002). These mutations include point mutations, deletions, duplication and insertions (Penta et al., 2001). Unfortunately, cellular consequences of depletion and mutations in the mitochondrial genome are not clear. Our study demonstrates an 10-fold decrease in pro-oxidant (superoxide) levels in cells lacking the mitochondrial genome. This decrease in 0 cells is consistent with a similar decrease in the levels of superoxide in 0 yeast cells and in breast epithelial cells (Gross et al., 2002; A.K. Rasmussen et al., submitted for publication; Delsite et al., unpublished results). Superoxide production has been demonstrated at complex I and complex III (Cadenas et al., 1977; Turrens and Boveris, 1980; Turrens et al., 1985; Marchetti et al., 1996) of the oxidative phosphorylation pathway. The loss of multiple subunits of mitochondrial respiratory complexes that are encoded by the mitochondrial genome may apparently inhibit superoxide radical formation.
The decrease in superoxide levels in cells with impaired oxidative phosphorylation was not reflected in a concurrent decrease in hydrogen peroxide. It is likely that the dismutation of superoxide is not the primary source of H2O2 in these 0 cells. H2O2 is also generated by the deamination of biogenic amines by monoamine oxidases (Hauptmann et al., 1996; reviewed in Cadenas and Davies, 2000). A previous study has described an increase in the fluorescence of dichlorofluorescein in HeLa 0 cells by microscopy, however, this study did not quantify H2O2 production (Miranda et al., 1999). In contrast, the results in our study are quantitative and report the average intensity of 10 000 cells. In this context it is noteworthy that treatment of isolated mitochondria or HeLa + cells with a complex III inhibitor (antimycin A), but not with inhibitors of complex I or II (rotenone or thenoyltrifluoroacetone, respectively), results in an increase in H2O2 production (Garcia-Ruiz et al., 1995). It is conceivable that initial mitochondrial inhibition (by antimycin A) causes a transient increase in hydrogen peroxide level, but that the procedure used to isolate 0 cells allows cells to adapt after this initial increase in hydrogen peroxide.
Our study demonstrates that both + and 0 cells contain similar levels of total superoxide dismutase activity. This suggests that the lower level of superoxide observed in 0 cells is not due to an increase in its catabolism. While higher levels of superoxide are observed in + cells, superoxide may be sufficiently catabolized to hydrogen peroxide in both cell types. The undetectable levels of the cyanide-insensitive mitochondrial MnSOD in both + and 0 cells may be a characteristic of the parental HeLa cells used in these studies. It has been reported that HeLa cells contain lower levels of total SOD activity than does the melanoma cell line DND-1A (Porta et al., 1996). The observed decrease in catalase activity in the 0 cells could lead to oxidative damage to DNA, proteins and lipids. Our study revealed no significant differences between 0 and + cells in the oxidation of proteins (data not shown). Instead, our study suggests that lower catalase activity may cause lipid peroxidation and damage to DNA.
The increase in TBA-reactive substances provided evidence for increased lipid peroxidation in 0 cells with mitochondrial dysfunction. This increase in lipid peroxides occurs in the presence of decreased levels of superoxide radical. Interestingly, a decrease in the activities of complexes I, II and III induced by carbon tetrachloride has been shown to be associated with increased lipid peroxides (Padma and Setty, 1999). Lipid peroxidation could also be induced by ferric complexes with ATP or citrate (reviewed in Kowaltowski and Vercesi, 1999). Fe(II) citrate-induced mitochondrial lipid peroxidation is believed to occur independently of ROS derived from the respiratory chain (Kowaltowski and Vercesi, 1999).
Although no increase in hydrogen peroxide level is observed in 0 cells, our results show that 0 cells were more sensitive to DNA damage by hydrogen peroxide when compared with + cells. It is likely due to increased lipid peroxidation by hydrogen peroxide, for it has been shown that oxidant treatment causes increased lipid peroxidation that induces damage directly to DNA (Vaca and Harms-Ringdahl, 1988; Vaca et al., 1988; Ames and Gold, 1991). It is also conceivable that superoxide present in 0 cells interacts with nitric oxide to form peroxynitrite, which in turn forms highly reactive hydroxy radicals that damage DNA. Our data suggest that GPx activity is higher in 0 cells, suggesting that these cells can tolerate a threshold level of oxidative damage induced by endogenous lipid peroxidation due to impairment of mitochondrial function. However, when the threshold is challenged by an exogenous oxidant, increased GPx activity is perhaps not enough to protect 0 cells against oxidative damage. It is also possible that DNA repair is impaired due to mitochondrial loss of function and thus 0 cells show elevated levels of oxidative DNA damage.
In summary, we demonstrate that impairment of mitochondrial function leads to a decreased level of superoxide without a concurrent decrease in oxidation of hydrogen peroxide as measured with the dyes DHE and CM-H2DCFDA, respectively. We also demonstrate that mitochondrial impairment contributes to lipid peroxidation and damage to the nuclear genome. Our analysis provides evidence for a role of impaired DNA repair in the pathophysiology of acquired mitochondrial diseases due to cancer treatment. These studies also suggest that 0 cells may serve as a suitable model for the study of the molecular response to endogenous oxidative stress and analysis of mitochondria-mediated nuclear genome instability.
|
Acknowledgements |
|---|
We thank Ms Leslie Meszler for flow cytometry analysis as well as Anders Elleby Engell-Kofoed, Janus Wiese Christoffersen, Sofie Dalbros Andersen and Sascha Emilie Liberti for help with Comet assays. We also thank Ms Ling Li and Dr Doug Spitz for their help in antioxidant assays. This research was supported by grants from the National Institutes of Health (RO1-097714 and CA66081), American Heart Association grant 9939223N and the Danish Cancer Society, the Danish Research Council and the Carlsberg Foundation. Robert Delsite was supported in part by NIH Training Grant T32 CA0936 from the National Cancer Institute.
|
Notes |
|---|
6To whom correspondence should be addressed. Tel: +1 716 845 8017; Fax: +1 716 845 1047; Email: keshav.singh{at}roswellpark.org
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Ames,B.N. and Gold,L.S. (1991) Endogenous mutagens and the causes of aging and cancer. Mutat. Res., 250, 3–16.[Web of Science][Medline]
Ames,B.N., Shigenaga,M.K. and Hagen,T.M. (1993) Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA, 90, 7915–7922.
Anderson,S., Bankier,A.T., Barrell,B.G. et al. (1981) Sequence and organization of the human mitochondrial genome. Nature, 290, 457–465.[CrossRef][Medline]
Beers,R.F.,Jr and Sizer,I.W. (1952) A spectrophotometer method for measuring the breakdown of H2O2 by catalase. J. Biol. Chem., 195, 133–140.
Bowie,A.G., Moynagh,P.N. and O’Neill,L.A. (1997) Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells. J. Biol. Chem., 272, 25941–25950.
Breen,A.P. and Murphy,J.A. (1995) Reactions of oxyl radicals with DNA. Free Radic. Biol. Med., 18, 1033–1077.[CrossRef][Web of Science][Medline]
Cadenas,E. and Davies,K.J. (2000) Mitochondrial free radical generation, oxidative stress and aging. Free Radic. Biol. Med., 29, 222–230.[CrossRef][Web of Science][Medline]
Cadenas,E., Boveris,A., Ragan,C.I. and Stoppani,A.O. (1977) Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch. Biochem. Biophys., 180, 2482–2457.
Castilho,R.F., Kowaltowski,A.J., Meinicke,A.R. and Vercesi,A.E. (1995) Oxidative damage of mitochondria induced by Fe(II)citrate or t-butyl hydroperoxide in the presence of Ca2+: effect of coenzyme Q redox state. Free Radic. Biol. Med., 18, 55–59.[CrossRef][Web of Science][Medline]
Fliss,M.S., Usadel,H., Caballero,O.L., Wu,L., Buta,M.R., Eleff,S.M., Jen,J. and Sidransky,D. (2002) Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science, 287, 2017–2019.
Garcia-Ruiz,C., Colell,A., Morales,A., Kaplowitz,N. and Fernandez-Checa,J.C. (1995) Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes. Mol. Pharmacol., 48, 825–834.[Abstract]
Garfin,D.E. (1990) One-dimensional gel electrophoresis. In Deutscher,M.P. (Ed.), Methods in Enzymology, Vol. 182, Guide to Protein Purification. Academic Press, San Diego, CA, pp. 425–441.
Gross,A., Pilcher,K., Blachly-Dyson,E., Basso,E., Jockel,J., Bassik,M.C., Korsmeyer,S.J. and Forte,M. (2000) Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L). Mol. Cell. Biol., 20, 3125–3136.
Ha,H.C., Woster,P.M., Yager,J.D. and Casero,R.A.,Jr (1997) The role of polyamine catabolism in polyamine analogue-induced programmed cell death. Proc. Natl Acad. Sci. USA, 94, 11557–11562.
Halliwell,B. (1978) Biochemical mechanisms accounting for the toxic action of oxygen on living organisms: the key role of superoxide dismutase. Cell Biol. Int. Rep., 2, 113–128.[CrossRef][Web of Science][Medline]
Hauptmann,N., Grimsby,J., Shih,J.C. and Cadenas,E. (1996) The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA. Arch. Biochem. Biophys., 335, 295–304.[CrossRef][Web of Science][Medline]
Hockenbery,D.M., Oltvai,Z.N., Yin,X.M., Milliman,C.L. and Korsmeyer,S.J. (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell, 75, 241–251.[CrossRef][Web of Science][Medline]
Jain,R.G., Furfine,E.S., Pedneault,L., White,A.J. and Lenhard,M.J. (2001) Metabolic complications associated with antiretroviral therapy. Antiviral Res., 51, 151–177.[CrossRef][Web of Science][Medline]
Janero,D.R. (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol. Med., 9, 515–540.[CrossRef][Web of Science][Medline]
Jones,J.B., Song,J.J., Hempen,P.M., Parmigiani,G., Hruban,R.H. and Kern,S.E. (2001) Detection of mitochondrial DNA mutations in pancreatic cancer offers a "mass"-ive advantage over detection of nuclear DNA mutations. Cancer Res., 61, 1299–1304.
Kang,D., Takeshige,K., Sekiguchi,M. and Singh,K.K. (1998) An introduction. In Singh,K.K. (Ed.), Mitochondrial DNA Mutations in Aging, Disease and Cancer. Springer-Verlag, Berlin, Germany, pp. 1–11.
Kowaltowski,A.J. and Vercesi,A.E. (1999) Mitochondrial damage induced by conditions of oxidative stress. Free Radic. Biol. Med., 26, 463–471.[CrossRef][Web of Science][Medline]
Lawrence,R. and Burk,R. (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Commun., 71, 952–958.[CrossRef][Web of Science][Medline]
LeDoux,S.P., Driggers,W.J., Hollensworth,B.S. and Wilson,G.L. (1999) Repair of alkylation and oxidative damage in mitochondrial DNA. Mutat. Res., 434, 149–159.[Web of Science][Medline]
Lewis,W., Copeland,W.C. and Day,B.J. (2001) Mitochondrial dna depletion, oxidative stress and mutation: mechanisms of dysfunction from nucleoside reverse transcriptase inhibitors. Lab. Invest., 81, 777–790.[Web of Science][Medline]
Lu,C.Y., Lee,H.C., Fahn,H.J. and Wei,Y.H. (1999) Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin. Mutat. Res., 423, 11–21.[Web of Science][Medline]
Marchetti,P., Susin,S.A., Decaudin,D., Gamen,S., Castedo,M., Hirsch,T., Zamzami,N., Naval,J., Senik,A. and Kroemer,G. (1996) Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res., 56, 2033–2038.
Mathupala,S.P., Rempel,A. and Pedersen,P.L. (1997) Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational and mutational events that lead to a critical role for type II hexokinase. J. Bioenerg. Biomembr., 29, 339–343.[CrossRef][Web of Science][Medline]
McCord,J.M. and Fridovich,I. (1970) The utility of superoxide dismutase in studying free radical reactions. II. The mechanism of the mediation of cytochrome c reduction by a variety of electron carriers. J. Biol. Chem., 245, 1374–1377.
McNamee,J.P., McLean,J.R., Ferrarotto,C.L. and Bellier,P.V. (1995) Comet assay: rapid processing of multiple samples. Mutat. Res., 466, 63–69.
Miranda,S., Foncea,R., Guerrero,J. and Leighton,F. (1999) Oxidative stress and upregulation of mitochondrial biogenesis genes in mitochondrial DNA-depleted HeLa cells. Biochem. Biophys. Res. Commun., 258, 44–49.[CrossRef][Web of Science][Medline]
Morais,R., Zinkewich-Peotti,K., Parent,M., Wang,H., Babai,F. and Zollinger,M. (1994) Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA. Cancer Res., 54, 3889–3896.
Nemoto,S., Takeda,K., Yu,Z.X., Ferrans,V.J. and Finkel,T. (2000) Role for mitochondrial oxidants as regulators of cellular metabolism. Mol. Cell. Biol., 20, 7311–7318.
Nishikawa,M., Nishiguchi,S., Shiomi,S., Tamori,A., Koh,N., Takeda,T., Kubo,S., Hirohashi,K., Kinoshita,H., Sato,E. and Inoue,M. (2000) Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma. Cancer Res., 61, 1843–1845.
Ochoa,J.J., Vilchez,M.J., Palacios,M.A., Garcia,J.J., Reiter,R.J. and Munoz-Hoyos,A. (2003) Melatonin protects against lipid peroxidation and membrane rigidity in erythrocytes from patients undergoing cardiopulmonary bypass surgery. J. Pineal Res., 35, 104–108.[CrossRef][Web of Science][Medline]
Padma,P. and Setty,O.H. (1999) Protective effect of Phyllanthus fraternus against carbon tetrachloride-induced mitochondrial dysfunction, Life Sci., 64, 2411–2417.[CrossRef][Web of Science][Medline]
Patockova,J., Krsiak,M., Marhol,P. and Tumova,E. (2003) Cerebrolysin inhibits lipid peroxidation induced by insulin hypoglycemia in the brain and heart of mice. Physiol. Res., 52, 455–460.[Web of Science][Medline]
Penta,J.A., Johnson,F.M., Wachsman,J.T. and Copeland,W.C. (2001) Mitochondrial DNA in human malignancy. Mutat. Res., 488, 119–133.[CrossRef][Web of Science][Medline]
Polyak,K., Li,Y., Zhu,H., Lengauer,C., Willson,J.K., Markowitz,S.D., Trush,M.A., Kinzler,K. and Vogelstein,B. (1998) Somatic mutations of the mitochondrial genome in human colorectal tumours. Nature Genet., 20, 291–293.[CrossRef][Web of Science][Medline]
Porta,C., Moroni,M., Guallini,P., Torri,C. and Marzatico,F. (1996) Antioxidant enzymatic system and free radicals pathway in two different human cancer cell lines. Anticancer Res., 16, 2741–2747.[Web of Science][Medline]
Rowe,T.C., Weissig,V. and Lawrence,J.W. (2001) Mitochondrial DNA metabolism targeting drugs. Adv. Drug Deliv. Rev., 49, 175–187.[CrossRef][Web of Science][Medline]
Sheu,J.Y., Chen,P.H., Tseng,W.C., Chen,C.Y., Tsai,L.Y. and Huang,Y.L. (2003) Spectrophotometric determination of a thiobarbituric acid-reactive substance in human hair. Anal. Sci., 19, 957–960.[CrossRef][Web of Science][Medline]
Shigenaga,M.K., Hagen,T.M. and Ames,B.N. (1994) Oxidative damage and mitochondrial decay in aging. Proc. Natl Acad. Sci. USA, 91, 10771–10778.
Singh,K.K., Russell,J., Sigala,B., Zhang,Y., Williams,J. and Keshav,K.F. (1999) Mitochondrial DNA determines the cellular response to cancer therapeutic agents. Oncogene, 18, 6641–6646.[CrossRef][Web of Science][Medline]
Spitz,D.R. and Oberley,L.W. (1989) An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem., 179, 8–18.[CrossRef][Web of Science][Medline]
Turrens,J.F. and Boveris,A. (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J., 191, 421–427.[Web of Science][Medline]
Turrens,J.F., Alexandre,A. and Lehninger,A.L. (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch. Biochem. Biophys., 237, 408–414.[CrossRef][Web of Science][Medline]
Vaca,C.E. and Harms-Ringdahl,M. (1988) Interaction of lipid peroxidation products with DNA: a review. Mutat. Res., 195, 137–149.[Web of Science][Medline]
Vaca,C.E., Wilhelm,J. and Harms-Ringdahl,M. (1988) Studies on lipid peroxidation in rat lever nuclei and isolated nuclear membrane. Biochim. Biophys. Acta, 958, 375–387.[Medline]
VandenHoek,T.L., Li,C., Shao,Z., Schumacker,P.T. and Becker,L.B. (1997) Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J. Mol. Cell. Cardiol., 29, 2571–2583.[CrossRef][Web of Science][Medline]
Wallace,D.C. (2001) A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found. Symp., 235, 247–263.[Medline]
Wallace,D.C., Lott,M.T., Brown,M.D., Huoponen,K. and Torroni,A.A.J. (1995) Report of the committee on human mitochondrial DNA. Cuticchia,A.J. (Ed.), Human Gene Mapping 1995: A Compendium. Johns Hopkins University Press, Baltimore, MD, pp. 910–954.
Wei,Y.H., Lu,C.Y., Wei,C.Y., Ma,Y.S. and Lee,H.C. (2001) Oxidative stress in human aging and mitochondrial disease—consequences of defective mitochondrial respiration and impaired antioxidant enzyme system. Chin. J. Physiol., 44, 1–11.[Web of Science][Medline]
White,A.J. (2001) Mitochondrial toxicity and HIV therapy. Sex. Transm. Infect., 77, 158–173.
Yakes,F.M. and Van Houten,B. (1997) Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl Acad. Sci. USA, 94, 514–519.
Received on January 13, 2003; accepted on August 14, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. A. Partridge, S. X.L. Huang, E. Hernandez-Rosa, M. M. Davidson, and T. K. Hei Arsenic Induced Mitochondrial DNA Damage and Altered Mitochondrial Oxidative Function: Implications for Genotoxic Mechanisms in Mammalian Cells Cancer Res., June 1, 2007; 67(11): 5239 - 5247. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||