Mitochondrial DNA damage via augmented oxidative stress regulates endoplasmic reticulum stress and autophagy: crosstalk, links and signaling.

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.

Alteration of mitochondrial function and insulin sensitivity in primary mouse skeletal muscle cells isolated from transgenic and knockout mice: role of ogg1.

Recent evidence has linked mitochondrial dysfunction and DNA damage, increased oxidative stress in skeletal muscle, and insulin resistance (IR). The purpose of this study was to determine the role of the DNA repair enzyme, human 8-oxoguanine DNA glycosylase/apurinic/apyrimidinic lyase (hOGG1), on palmitate-induced mitochondrial dysfunction and IR in primary cultures of skeletal muscle derived from hind limb of ogg1(-/-) knockout mice and transgenic mice, which overexpress human (hOGG1) in mitochondria (transgenic [Tg]/MTS-hOGG1). Following exposure to palmitate, we evaluated mitochondrial DNA (mtDNA) damage, mitochondrial function, production of mitochondrial reactive oxygen species (mtROS), mitochondrial mass, JNK activation, insulin signaling pathways, and glucose uptake. Palmitate-induced mtDNA damage, mtROS, mitochondrial dysfunction, and activation of JNK were all diminished, whereas ATP levels, mitochondrial mass, insulin-stimulated phosphorylation of Akt (Ser 473), and insulin sensitivity were increased in primary myotubes isolated from Tg/MTS-hOGG1 mice compared to myotubes isolated from either knockout or wild-type mice. In addition, both basal and maximal respiratory rates during mitochondrial oxidation on pyruvate showed a variable response, with some animals displaying an increased respiration in muscle fibers isolated from the transgenic mice. Our results support the model that DNA repair enzyme OGG1 plays a pivotal role in repairing mtDNA damage, and consequently, in mtROS production and regulating downstream events leading to IR in skeletal muscle.

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism.

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Overcoming trastuzumab resistance in breast cancer by targeting dysregulated glucose metabolism.

Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.

Emerging metabolic targets in cancer therapy.

Cancer cells are different from normal cells in their metabolic properties. Normal cells mostly rely on mitochondrial oxidative phosphorylation to produce energy. In contrast, cancer cells depend mostly on glycolysis, the aerobic breakdown of glucose into ATP. This altered energy dependency is known as the "Warburg effect" and is a hallmark of cancer cells. In recent years, investigating the metabolic changes within cancer cells has been a rapidly growing area. Emerging evidence shows that oncogenes that drive the cancer-promoting signals also drive the altered metabolism. Although the exact mechanisms underlying the Warburg effect are unclear, the existing evidence suggests that increased glycolysis plays an important role in support malignant behavior of cancer cells. A thorough understanding of the unique metabolism of cancer cells will help to design of more effective drugs targeting metabolic pathways, which will greatly impact the capacity to effectively treat cancer patients. Here we provide an overview of the current understanding of the Warburg effect upon tumor cell growth and survival, and discussion on the potential metabolic targets for cancer therapy.

The approaches for manipulating mitochondrial proteome.

Over the past decade a large volume of research data has accumulated which has established a fundamental role for mitochondria in normal cellular functioning, as well as in various pathologies. Mitochondria play a pivotal role in metabolism and energy production, and are one of the key players involved in programmed cell death. On the other hand, mitochondrial dysfunction is implicated, directly or indirectly in numerous pathological conditions including inherited mitochondrial disorders, diabetes, cardiovascular and neurodegenerative diseases, and a variety of malignancies. The ability to modulate mitochondrial function by altering the diverse protein component of this organelle may be of great value for developing future therapeutic interventions. This review will discuss approaches used to introduce proteins into mitochondria. One group of methods utilizes strategies aimed at expressing proteins from genes in the nucleus. These include overexpression of nuclear-encoded mitochondrial proteins, allotopic expression, which is the re-coding and relocation of mitochondrial genes to the nucleus for expression and subsequent delivery of their gene products to mitochondria, and xenotopic expression, which is the nuclear expression of genes coding electron transport chain components from distant species, for delivery of their products to mammalian mitochondria. Additionally, antigenomic and progenomic strategies which focus on expression of mitochondrially targeted nuclear proteins involved in the maintenance of mtDNA will be discussed. The second group of methods considered will focus on attempts to use purified proteins for mitochondrial delivery. Special consideration has been given to the complexities involved in targeting exogenous proteins to mitochondria.

Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol.

BACKGROUND: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. RESULTS: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. CONCLUSION: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Mitochondrial DNA damage initiates a cell cycle arrest by a Chk2-associated mechanism in mammalian cells.

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes.

Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARgamma antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARgamma-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

Targeting repair proteins to the mitochondria of mammalian cells through stable transfection, transient transfection, viral transduction, and TAT-mediated protein transduction.

The mitochondrial genome represents a target for exogenous and endogenous damage. Its necessity for successful electron transport makes its repair valuable to the cell. Previous work from our lab has shown that mitochondrial DNA (mtDNA) can be repaired in mammalian cells, and the use of mitochondrial-targeted repair proteins can augment repair to enhance viability following genotoxic stress. In addition, it has also been shown that other repair enzymes that are targeted to the mitochondria can sensitize the cell to DNA damaging agents, thereby aiding the effectiveness of certain chemotherapeutic agents. The methods herein describe the development of mitochondrial-targeted proteins using plasmids or protein transduction domains. It includes the utilization of these constructs to create stably transfected cell lines, transiently transfected cell lines, viral-mediated transduction, and protein transduction domain-mediated mitochondrial protein localization. The end result will be a mammalian cell that expresses the mitochondrial-targeted protein of interest.

Mitochondrial DNA repair in aging and disease.

Mitochondria are organelles which, according to the endosymbiosis theory, evolved from purpurbacteria approximately 1.5 billion years ago. One of the unique features of mitochondria is that they have their own genome. Mitochondria replicate and transcribe their DNA semiautonomously. Like nuclear DNA, mitochondrial DNA (mtDNA) is constantly exposed to DNA damaging agents. Regarding the repair of mtDNA, the prevailing concept for many years was that mtDNA molecules suffering an excess of damage would simply be degraded to be replaced by newly generated successors copied from undamaged genomes. However, evidence now clearly shows that mitochondria contain the machinery to repair the damage to their genomes caused by certain endogenous or exogenous damaging agents. The link between mtDNA damage and repair to aging, neurodegeneration, and carcinogenesis-associated processes is the subject of this review.

Altering DNA base excision repair: use of nuclear and mitochondrial-targeted N-methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents.

Primary astrocyte cultures were used to investigate the modulation of DNA repair as a tool for sensitizing astrocytes to genotoxic agents. Base excision repair (BER) is the principal mechanism by which mammalian cells repair alkylation damage to DNA and involves the processing of relatively nontoxic DNA adducts through a series of cytotoxic intermediates during the course of restoring normal DNA integrity. An adenoviral expression system was employed to target high levels of the BER pathway initiator, N-methylpurine glycosylase (MPG), to either the mitochondria or nucleus of primary astrocytes to test the hypothesis that an alteration in BER results in increased alkylation sensitivity. Increasing MPG activity significantly increased BER kinetics in both the mitochondria and nuclei. Although modulating MPG activity in mitochondria appeared to have little effect on alkylation sensitivity, increased nuclear MPG activity resulted in cell death in astrocyte cultures treated with methylnitrosourea (MNU). Caspase-3 cleavage was not detected, thus indicating that these alkylation sensitive astrocytes do not undergo a typical programmed cell death in response to MNU. Astrocytes were found to express relatively high levels of antiapoptotic Bcl-2 and Bcl-XL and very low levels of proapoptotic Bad and Bid suggesting that the mitochondrial pathway of apoptosis may be blocked making astrocytes less vulnerable to proapoptotic stimuli compared with other cell types. Consequently, this unique characteristic of astrocytes may be responsible, in part, for resistance of astrocytomas to chemotherapeutic agents.

Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress.

Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.

Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis.

An increasing body of evidence suggests that nitric oxide (NO) can be cytotoxic and induce apoptosis. NO can also be genotoxic and cause DNA damage and mutations. It has been shown that NO damages mitochondrial DNA (mtDNA) to a greater extent than nuclear DNA. Previously, we reported that conditional targeting of the DNA repair protein hOGG1 into mitochondria using a mitochondria targeting sequence (MTS) augmented mtDNA repair of oxidative damage and enhanced cellular survival. To determine whether enhanced repair resulting from augmented expression of hOGG1 could also protect against the deleterious effects of NO, we used HeLa TetOff/MTS-OGG1-transfected cells to conditionally express hOGG1 in mitochondria. The effects of additional hOGG1 expression on repair of NO-induced mtDNA damage and cell survival were evaluated. These cells, along with vector transfectants, in either the presence or absence of doxycycline (Dox), were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate). Functional studies revealed that cells expressing recombinant hOGG1 were more proficient at repairing NO-induced mtDNA damage, which led to increased cellular survival following NO exposure. Moreover, the results described here show that conditional expression of hOGG1 in mitochondria decreases NO-induced inhibition of ATP production and protects cells from NO-induced apoptosis.

Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria.

Chronic exposure to elevated levels of free fatty acids (FFAs) impairs pancreatic beta-cell function and contributes to the decline of insulin secretion in type 2 diabetes. Previously, we reported that FFAs caused increased nitric oxide (NO) production, which damaged mitochondrial DNA (mtDNA) and ultimately led to apoptosis in INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase (hOGG1) downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase. Successful integration of MTS-OGG1 into the INS-1 cellular genome was confirmed by Southern blot analysis. Western blots and enzyme activity assays revealed that hOGG1 was targeted to mitochondria and the recombinant enzyme was active. MTS-OGG1 cells showed a significant decrease in FFA-induced mtDNA damage compared with vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced inhibition of ATP production and protected INS-1 cells from apoptosis. These results indicate that mtDNA damage plays a pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore, targeting DNA repair enzymes into beta-cell mitochondria could be a potential therapeutic strategy for preventing or delaying the onset of type 2 diabetes symptoms.

Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance.

Neurodegeneration can occur as a result of endogenous oxidative stress. Primary cerebellar granule cells were used in this study to determine if mitochondrial DNA (mtDNA) repair deficiencies correlate with oxidative stress-induced apoptosis in neuronal cells. Granule cells exhibited a significantly higher intracellular oxidative state compared with primary astrocytes as well as increases in reductants, such as glutathione, and redox sensitive signaling molecules, such as AP endonuclease/redox effector factor-1. Cerebellar granule cultures also exhibited an increased susceptibility to exogenous oxidative stress. Menadione (50 muM) produced twice as many lesions in granule cell mtDNA compared with astrocytes, and granule cell mtDNA repair was significantly less efficient. A decreased capacity to repair oxidative mtDNA damage correlates strongly with mitochondrial initiated apoptosis in these neuronal cultures. Interestingly, the mitochondrial activities of initiators for base excision repair (BER), the bifunctional glycosylase/AP lyases as well as AP endonuclease, were significantly higher in cerebellar granule cells compared with astrocytes. The increased mitochondrial AP endonuclease activity in combination with decreased polymerase gamma activity may cause an imbalance in oxidative BER leading to an increased production and persistence of mtDNA damage in neurons when treated with menadione. This study provides evidence linking neuronal mtDNA repair capacity with oxidative stress-related neurodegeneration.

Cytokines induce nitric oxide-mediated mtDNA damage and apoptosis in oligodendrocytes. Protective role of targeting 8-oxoguanine glycosylase to mitochondria.

Nitric oxide (NO) that is produced by inducible NO synthase (iNOS) in glial cells is thought to contribute significantly to the pathogenesis of multiple sclerosis. Oligodendrocytes can be stimulated to express iNOS by inflammatory cytokines, which are known to accumulate in the multiple sclerotic brain. The potentially pathological levels of NO produced under these circumstances can target a wide spectrum of intracellular components. We hypothesized that one of the critical targets for damage that leads to disease is mtDNA. In this study, we found that cytokines, in particular a combination of tumor necrosis factor-alpha (50 ng/ml) and IFNgamma (25 ng/ml), cause elevated NO production in primary cultures of rat oligodendrocytes. Western blot analysis revealed a strong enhancement of iNOS expression 48 h after cytokine treatment. Within the same time period, NO-mediated mtDNA damage was shown by Southern blot analysis and by ligation-mediated PCR. Targeting the DNA repair enzyme human 8-oxoguanine DNA glycosylase (hOGG1) to the mitochondria of oligodendrocytes had a protective effect against this cytokine-mediated mtDNA damage. Moreover, it was shown that mitochondrial transport sequence hOGG1-transfected oligodendrocytes had fewer apoptotic cells compared with cells containing vector only following treatment with the cytokines. Subsequent experiments revealed that targeting hOGG1 to mitochondria reduces the activation of caspase-9, showing that this recombinant protein works to reduce apoptosis that is occurring through a mitochondria-based pathway.

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells.

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.

Involvement of mtDNA damage in free fatty acid-induced apoptosis.

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.