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.     

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.     

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.     

Anin VitroModel of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In anin vitromodel, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similarin vitromodels.     

Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide

The effect of chitosan oligosaccharide (COS, 1 kDa 相似文献   

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.     

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.     

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.     

The fate of damaged mitochondrial DNA in the cell

Mitochondrion is a double membrane organelle that is responsible for cellular respiration and production of most of the ATP in eukaryotic cells. Mitochondrial DNA (mtDNA) is the genetic material carried by mitochondria, which encodes some essential subunits of respiratory complexes independent of nuclear DNA. Normally, mtDNA binds to certain proteins to form a nucleoid that is stable in mitochondria. Nevertheless, a variety of physiological or pathological stresses can cause mtDNA damage, and the accumulation of damaged mtDNA in mitochondria leads to mitochondrial dysfunction, which triggers the occurrence of mitochondrial diseases in vivo. In response to mtDNA damage, cell initiates multiple pathways including mtDNA repair, degradation, clearance and release, to recover mtDNA, and maintain mitochondrial quality and cell homeostasis. In this review, we provide our current understanding of the fate of damaged mtDNA, focus on the pathways and mechanisms of removing damaged mtDNA in the cell.     

Cytokine- and Endotoxin-Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine- and LPS-stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin-1β (IL-1β; 10 ng/ml, 24 h), interferon-γ (IFN-γ; 100 U/ml, 24 h), or tumor necrosis factor-α (TNF-α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL-1β, and TNF-α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN-γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.     

The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress

The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.     

The role of nitric oxide in the pathogenesis of multiple sclerosis

Nitric oxide is a free radical gas, NO, of paramount relevance in biology. The enzymes responsible for the synthesis of NO from L-arginine in mammalian tissues are known as nitric oxide synthases (NOS). The inducible NOS (iNOS) is associated with the development of a number of autoimmune diseases. iNOS is induced on monocytes, cells playing a key role in the initiation and progression of the immune response. Induction of the enzyme is effected by proinflammatory cytokines, immunomodulating peptides, and even beta-endorphin through a mechanism involving an increase in cAMP. An excessive production of NO has been implicated in the severe lesions observed in multiple sclerosis (MS). Nitrosation of proteins caused by NO in monocytes may contribute to the formation of new epitopes involved in the autoimmune response. Monocytes/macrophages enhance also their cytotoxic capacity through an increase in NO. iNOS seems to establish a link between neuroendocrine and immune system through beta-endorphin explaining stress-related relapses in MS. One of the causes of demyelination is the lysis of oligodendrocytes by cytotoxic T lymphocytes (CTLs); and T cell response is also known to be modulated by NO.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.     

The amount and integrity of mtDNA in maize decline with development

In maize and other grasses there is a developmental gradient from the meristematic cells at the base of the stalk to the differentiated cells at the leaf tip. This gradient presents an opportunity to investigate changes in mitochondrial DNA (mtDNA) that accompany growth under light and dark conditions, as done previously for plastid DNA. Maize mtDNA was analyzed by DAPI-DNA staining of individual mitochondria, gel electrophoresis/blot hybridization, and real-time qPCR. Both the amount and integrity of the mtDNA were found to decline with development. There was a 20-fold decline in mtDNA copy number per cell from the embryo to the light-grown leaf blade. The amount of DNA per mitochondrial particle was greater in dark-grown leaf blade (24 copies, on average) than in the light (2 copies), with some mitochondria lacking any detectable DNA. Three factors that influence the demise of mtDNA during development are considered: (1) the decision to either repair or degrade mtDNA molecules that are damaged by the reactive oxygen species produced as byproducts of respiration; (2) the generation of ATP by photophosphorylation in chloroplasts, reducing the need for respiratory-competent mitochondria; and (3) the shift in mitochondrial function from energy-generating respiration to photorespiration during the transition from non-green to green tissue.     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.