Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

DNA repair, mitochondria, and neurodegeneration.

Accumulation of nuclear and mitochondrial DNA damage is thought to be particularly deleterious in post-mitotic cells, which cannot be replaced through cell division. Recent experimental evidence demonstrates the importance of DNA damage responses for neuronal survival. Here, we summarize current literature on DNA damage responses in the mammalian CNS in aging and neurodegeneration. Base excision repair (BER) is the main pathway for the removal of small DNA base modifications, such as alkylation, deamination and oxidation, which are generated as by-products of normal metabolism and accumulate with age in various experimental models. Using neuronal cell cultures, human brain tissue and animal models, we and others have shown an active BER pathway functioning in the brain, both in the mitochondrial and nuclear compartments. Mitochondrial DNA repair may play a more essential role in neuronal cells because these cells depend largely on intact mitochondrial function for energy metabolism. We have characterized several BER enzymes in mammalian mitochondria and have shown that BER activities change with age in mitochondria from different brain regions. Together, the results reviewed here advocate that mitochondrial DNA damage response plays an important role in aging and in the pathogenesis of neurodegenerative diseases.

No evidence of mitochondrial respiratory dysfunction in OGG1-null mice deficient in removal of 8-oxodeoxyguanine from mitochondrial DNA.

Accumulation of high levels of mutagenic oxidative mitochondrial DNA (mtDNA) lesions like 8-oxodeoxyguanine (8-oxodG) is thought to be involved in the development of mitochondrial dysfunction in aging and in disorders associated with aging. Mice null for oxoguanine DNA glycosylase (OGG1) are deficient in 8-oxodG removal and accumulate 8-oxodG in mtDNA to levels 20-fold higher than in wild-type mice (N.C. Souza-Pinto et al., 2001, Cancer Res. 61, 5378-5381). We have used these animals to investigate the effects on mitochondrial function of accumulating this particular oxidative base modification. Despite the presence of high levels of 8-oxodG, mitochondria isolated from livers and hearts of Ogg1-/- mice were functionally normal. No differences were detected in maximal (chemically uncoupled) respiration rates, ADP phosphorylating respiration rates, or nonphosphorylating rates with glutamate/malate or with succinate/rotenone. Similarly, maximal activities of respiratory complexes I and IV from liver and heart were not different between wild-type and Ogg1-/- mice. In addition, there was no indication of increased oxidative stress in mitochondria from Ogg1-/- mice, as measured by mitochondrial protein carbonyl content. We conclude, therefore, that highly elevated levels of 8-oxodG in mtDNA do not cause mitochondrial respiratory dysfunction in mice.

The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity.

The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; alpha-Ogg1, which mainly localizes to the nucleus and beta-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from rho(0) cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that beta-Ogg1 protein levels are approximately 80% reduced in rho(0) cells, suggesting beta-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant beta-Ogg1. Surprisingly, recombinant beta-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since beta-Ogg1 lacks the C-terminal alphaO helix present in alpha-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317-323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the alphaO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why beta-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of alpha-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that alpha-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. beta-Ogg1 may play a novel role in human mitochondria.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial dna of OGG1-defective mice.

Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demonstrated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1(-/-) mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion. Consistent with an important role for the OGG1 protein in the removal of 8-oxodG from the mitochondrial genome, we found that mtDNA isolated from liver from OGG1-null mutant animals contained 20-fold more 8-oxodG than mtDNA from wild-type animals.

DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts.

8-oxo-deoxyguanosine (8-oxodG) is one of the major DNA lesions formed upon oxidative attack of DNA. It is a mutagenic adduct that has been associated with pathological states such as cancer and aging. Base excision repair (BER) is the main pathway for the repair of 8-oxodG. There is a great deal of interest in the question about age-associated accumulation of this DNA lesion and its intracellular distribution, particularly with respect to mitochondrial or nuclear localization. We have previously shown that 8-oxodG-incision activity increases with age in rat mitochondria obtained from both liver and heart. In this study, we have investigated the age-associated changes in DNA repair activities in both mitochondrial and nuclear extracts obtained from mouse liver. We observed that 8-oxodG incision activity of mitochondrial extracts increases significantly with age, from 13.4 + or - 2.2 fmoles of oligomer/100 microg of protein/16 h at 6 to 18.6 + or - 4.9 at 14 and 23.7 + or - 3.8 at 23 months of age. In contrast, the nuclear 8-oxodG incision activity showed no significant change with age, and in fact slightly decreased from 11.8 + or - 3 fmoles/50 microg of protein/2 h at 6 months to 9.7 + or - 0.8 at 14 months. Uracil DNA glycosylase and endonuclease G activities did not change with age in nucleus or mitochondria. Our results show that the repair of 8-oxodG is regulated differently in nucleus and mitochondria during the aging process. The specific increase in 8-oxodG-incision activity in mitochondria, rather than a general up-regulation of DNA metabolizing enzymes in those organelles, suggests that this pathway may be up regulated during aging in mice.

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Mechanism of tetrahydroxy-1,4-quinone cytotoxicity: involvement of CA2+ and H2O2 in the impairment of DNA replication and mitochondrial function.

In this work we investigated the toxicity of a polyphenolic p-benzoquinone derivative, the tetrahydroxy-1,4-quinone (THQ) toward V79 Chinese hamster fibroblasts and analyzed the role of H2O2 and Ca2+ in that mechanism. The exposure of exponentially growing cultures to THQ, in the presence of 1.0 mM Ca2+, caused a dose-dependent inhibition of cell growth and DNA synthesis. Complete prevention of those effects by catalase indicated that H2O2-induced damages should underlie both toxic processes. Further detection of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in cells exposed to THQ plus Ca2+, together with the partial protection conferred by the intracellular Ca(2+)-chelator fura-2 against cell growth inhibition, indicated that a disruption of Ca2+ homeostasis is a determinant event in THQ cytotoxicity. Furthermore, the intracellular accumulation of rhodizonic acid (RDZ), the primary oxidative product of THQ, indicated that THQ, or its corresponding semiquinone form, was entering the cells and undergoing further autoxidation to RDZ. It was also evidenced that mitochondria represent an important target in the development of THQ toxicity, as shown by the disruption of the transmembrane electrical potential (delta psi) of isolated rat liver mitochondria, as well as by the Ca(2+)-release by mitochondria of permeabilized V79 cells. We concluded that disruption of Ca2+ homeostasis and generation of H2O2 are critically involved in THQ-induced impairment of DNA replication and mitochondrial functions, leading ultimately to cell growth inhibition.