Palmitate induced mitochondrial deoxyribonucleic acid damage and apoptosis in l6 rat skeletal muscle cells.

A major characteristic of type 2 diabetes mellitus (T2DM) is insulin resistance in skeletal muscle. A growing body of evidence indicates that oxidative stress that results from increased production of reactive oxygen species and/or reactive nitrogen species leads to insulin resistance, tissue damage, and other complications observed in T2DM. It has been suggested that muscular free fatty acid accumulation might be responsible for the mitochondrial dysfunction and insulin resistance seen in T2DM, although the mechanisms by which increased levels of free fatty acid lead to insulin resistance are not well understood. To help resolve this situation, we report that saturated fatty acid palmitate stimulated the expression of inducible nitric oxide (NO) synthase and the production of reactive oxygen species and NO in L6 myotubes. Additionally, palmitate caused a significant dose-dependent increase in mitochondrial DNA (mtDNA) damage and a subsequent decrease in L6 myotube viability and ATP levels at concentrations as low as 0.5 mM. Furthermore, palmitate induced apoptosis, which was detected by DNA fragmentation, caspase-3 cleavage, and cytochrome c release. N-acetyl cysteine, a precursor compound for glutathione formation, aminoguanidine, an inducible NO synthase inhibitor, and 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III), a peroxynitrite inhibitor, all prevented palmitate-induced mtDNA damage and diminished palmitate-induced cytotoxicity. We conclude that exposure of L6 myotubes to palmitate induced mtDNA damage and triggered mitochondrial dysfunction, which caused apoptosis. Additionally, our findings indicate that palmitate-induced mtDNA damage and cytotoxicity in skeletal muscle cells were caused by overproduction of peroxynitrite.

Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults.

Cells of the CNS are constantly exposed to agents which damage DNA. Although much attention has been paid to the effects of this damage on nuclear DNA, the nucleus is not the only organelle containing DNA. Within each cell, there are hundreds to thousands of mitochondria. Within each mitochondrion are multiple copies of the mitochondrial genome. These genomes are extremely vulnerable to insult and mutations in mitochondrial DNA (mtDNA) have been linked to several neurodegenerative diseases, as well as the normal process of aging. The principal mechanism utilized by cells to avoid DNA mutations is DNA repair. Multiple pathways of DNA repair have been elucidated for nuclear DNA. However, it appears that only base excision repair is functioning in mitochondria. This repair pathway is responsible for the removal of most endogenous damage including alkylation damage, depurination reactions and oxidative damage. Within the rat CNS, there are cell-specific differences mtDNA repair. Astrocytes exhibit efficient repair, whereas, other glial cell types and neuronal cells exhibit a reduced ability to remove lesions from mtDNA. Additionally, a correlation was observed between those cells with reduced mtDNA repair and an increase in the induction of apoptosis. To demonstrate a causative relationship, a strategy of targeting DNA repair proteins to mitochondria to enhance mtDNA repair capacity was employed. Enhancement of mtDNA repair in oligodendrocytes provided protection from reactive oxygen species- and cytokine-induced apoptosis. These experiments provide a novel strategy for protecting sensitive CNS cells from genotoxic insults and thus provide new treatment options for neurodegenerative diseases.

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Base excision repair of mitochondrial DNA damage in mammalian cells.

This review of the work from our laboratory describes initial studies in which base excision repair in mtDNA was first seen. It considers the results of experiments in which the substrates for mtDNA repair were identified. The discussion then focuses on studies during which the sequence context for mtDNA damage and repair were explored. Next, it addresses factors that have been identified that influence mtDNA repair. Finally, it summarizes the results of studies that evaluated cell-specific differences in the repair of mtDNA and explored some of the biological consequences of these differences.

Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.

Poly(ADP-ribose) polymerase facilitates the repair of N-methylpurines in mitochondrial DNA.

This study was designed to test the hypothesis that poly(ADP-ribose) polymerase (PARP) plays a role in the repair of damage to mitochondrial DNA (mtDNA). A rat insulinoma cell line was transfected with a PARP antisense vector that was under the control of a dexamethasone promoter. Transfected cells were selected for stable integration of the antisense vector. Several cell lines containing the antisense vector were isolated. For these studies, one of these lines (clone 5) was chosen for further evaluation. When cells were treated with dexamethasone for 72 h, PARP activity was diminished by 60%. Western blot analysis revealed a concomitant reduction in PARP protein. When clone 5 cells were exposed to the simple methylating agent methylnitrosourea (MNU) without previous treatment with dexamethasone, repair of lesions in mtDNA was found to be similar to that seen in wild-type cells or in wild-type cells treated with dexamethasone. However, when clone 5 cells were pretreated with dexamethasone for 72 h, repair of MNU-induced damage was significantly inhibited. To ascertain whether the PARP activity that was inhibited by the antisense treatment was the same as that found in the nucleus, repair studies were performed on fibroblasts derived from PARP knockout mice and their normal wild-type controls. Attenuated repair was also seen in the cells in which the gene for PARP was inactivated. These are the first studies to demonstrate that PARP can facilitate the repair of simple alkylation damage to mtDNA.

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.

Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria.

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

Glial cell type-specific responses to menadione-induced oxidative stress.

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.

Alzheimer beta protein mediated oxidative damage of mitochondrial DNA: prevention by melatonin.

Most contemporary progress in Alzheimer's disease (AD) stems from the study of a 42 43 amino acid peptide. called the amyloid beta protein (Abeta), as the main neuropathologic marker of the disorder. It has been demonstrated that Abeta has neurotoxic properties and that such effects are mediated by free-radicals. Exposure of neuronal cells to Abeta results in a spectrum of oxidative lesions that are profoundly harmful to neuronal homeostasis. We had previously shown that Abeta25-35 induces oxidative damage to mitochondrial DNA (mtDNA) and that this modality of injury is prevented by melatonin. Because Abeta25 35 does not occur in AD and because the mode of toxicity by Abeta25-35 may be different from that of Abeta1-42 (the physiologically relevant form of Abeta), we extended our initial observations to determine whether oxidative damage to mtDNA could also be induced by Abeta1-42 and whether this type of injury is prevented by melatonin. Exposure of human neuroblastoma cells to Abeta1-42 resulted in marked oxidative damage to mtDNA as determined by a quantitative polymerase chain reaction method. Addition of melatonin to cell cultures along with Abeta completely prevented the damage. This study supports previous findings with Abeta25-35, including a causative role for Abeta in the mitochondrial oxidative lesions present in AD brains. Most important, the data confirms the neuroprotective role of melatonin in Abeta-mediated oxidative injury. Because melatonin also inhibits amyloid aggregation, lacks toxicity, and efficiently crosses the blood-brain barrier, this hormone appears superior to other available antioxidants as a candidate for pharmacologic intervention in AD.

Nitric oxide-induced damage to mtDNA and its subsequent repair.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.

Defective repair of oxidative damage in mitochondrial DNA in Down's syndrome.

Recent evidence indicates that oxidative DNA damage may be a major cause of aging. One of the more sensitive targets is the mitochondrial genome which is 10 times more susceptible to mutation than is the nuclear genome. A number of age-related neuromuscular degenerative diseases also have been associated with mutations in mitochondrial DNA (mtDNA), and progressive accumulation of oxidative damage in mtDNA from neuronal tissues over time has been shown. In support of the notion that oxidative stress leads to aging is the finding in Down's syndrome (DS), which is characterized by premature aging, that there is enhanced oxidative stress resulting from the aberrant expression of CuZn superoxide dismutase (CuZn SOD). On the basis of these observations, we hypothesized that there may be defective repair of oxidative damage in mtDNA which would ultimately lead to defective electron transport and concomitant enhanced production of reactive oxygen species (ROS). This effect would heighten the oxidative burden in the cell and accelerate the development of phenotypes associated with aging. To evaluate repair of oxidative damage in mtDNA, fibroblasts from several DS patients were treated with the reactive oxygen generator menadione. Oxidative damage was assessed at 0, 2, and 6 h after exposure using a Southern-blot technique and a mtDNA specific probe. The results of these studies show that DS cells are impaired in their ability to repair oxidative damage to mtDNA compared to age-matched control cells. Therefore, this data supports the possibility that increased production of ROS from mitochondria plays a crucial role in the development of aging phenotypes.

Glial cell-specific differences in response to alkylation damage.

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type-specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N-methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N-nitroso compounds in the environment or in chemotherapeutic regimen.

The amyloid beta protein induces oxidative damage of mitochondrial DNA.

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (A beta) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC 12 cells to an A beta fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 microM A beta (25-35) or to 50 microM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease III or FAPy glycosylase was used to detect pyrimidine or purine lesions, respectively. Cells exposed to A beta exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the A beta-induced oxidative damage to mtDNA. Mitochondrial dysfunction in AD has been demonstrated by several laboratories. This study provides experimental evidence supporting a causative role of A beta in mitochondrial lesions of AD.

Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA.

Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens. Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes. We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase. Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress. Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage. These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.

Mitochondrial DNA in beta-cells is a sensitive target for damage by nitric oxide.

Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to beta-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1beta) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, beta-cell cultures were treated with IL-1beta for 18 h. Other cultures were treated with IL-1beta and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1beta caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1beta and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA.

Repair of oxidative damage in nuclear DNA sequences with different transcriptional activities.

This study was designed to investigate the repair of oxidative damage in nuclear DNA sequences with different transcriptional activities. Chinese hamster ovary (CHO) cells were treated with the oxygen radical generator hypoxanthine/xanthine oxidase (Hyp/XO). Damage and repair were evaluated in 14-kb restriction fragments containing either the DHFR gene, a 3'-non-transcribed flanking region, or the c-fos gene using a quantitative Southern blot technique. Damage to the sugar-phosphate backbone and abasic sites were detected by measuring their lability in alkali conditions. Lesions in DNA bases were identified using the bacterial repair enzyme endonuclease III, which predominantly recognizes damage to thymines and cytosines, and formamidopyrimidine-DNA glycosylase, which recognizes 8-oxoguanine and purines with fractured imidazole rings. The results showed that similar amounts of all types of oxidative damage were produced in both the transcribed and non-transcribed sequences following a 1-h exposure to the radical generator. Repair in all sequences was rapid, with approximately 60% removal of lesions observed by 1 h. Therefore, within these sequences, the repair of oxidative lesions is much faster than that of other types of damage, such as those induced by alkylating toxins and UV irradiation, and the repair is not affected appreciably by transcriptional status.