A systematic evaluation of normalization methods and probe replicability using infinium EPIC methylation data.

BACKGROUND: The Infinium EPIC array measures the methylation status of > 850,000 CpG sites. The EPIC BeadChip uses a two-array design: Infinium Type I and Type II probes. These probe types exhibit different technical characteristics which may confound analyses. Numerous normalization and pre-processing methods have been developed to reduce probe type bias as well as other issues such as background and dye bias. METHODS: This study evaluates the performance of various normalization methods using 16 replicated samples and three metrics: absolute beta-value difference, overlap of non-replicated CpGs between replicate pairs, and effect on beta-value distributions. Additionally, we carried out Pearson's correlation and intraclass correlation coefficient (ICC) analyses using both raw and SeSAMe 2 normalized data. RESULTS: The method we define as SeSAMe 2, which consists of the application of the regular SeSAMe pipeline with an additional round of QC, pOOBAH masking, was found to be the best performing normalization method, while quantile-based methods were found to be the worst performing methods. Whole-array Pearson's correlations were found to be high. However, in agreement with previous studies, a substantial proportion of the probes on the EPIC array showed poor reproducibility (ICC < 0.50). The majority of poor performing probes have beta values close to either 0 or 1, and relatively low standard deviations. These results suggest that probe reliability is largely the result of limited biological variation rather than technical measurement variation. Importantly, normalizing the data with SeSAMe 2 dramatically improved ICC estimates, with the proportion of probes with ICC values > 0.50 increasing from 45.18% (raw data) to 61.35% (SeSAMe 2).

Increased H2O2 levels and p53 stabilization lead to mitochondrial dysfunction in XPC-deficient cells.

XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in Xeroderma pigmentosum complementation group C (XP-C) (XPC-null) cells compared with an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, whereas AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine increased glutathione concentration and decreased basal H2O2 production, p53 levels and sensitivity to AA treatment in the XPC-null back to the levels found in XPC-wt cells. Thus, the results suggest a critical role for mitochondrially generated H2O2 in the regulation of p53 expression, which in turn modulates XP-C sensitivity to agents that cause mitochondrial stress.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Mechanisms of manganese-induced neurotoxicity in primary neuronal cultures: the role of manganese speciation and cell type.

Manganese (Mn) is an essential trace element required for the proper functioning of a variety of physiological processes. However, chronic exposures to Mn can cause neurotoxicity in humans, especially when it occurs during critical stages of the central nervous system development. The mechanisms mediating this phenomenon as well as the contribution of Mn speciation and the sensitivity of different types of neuronal cells in such toxicity are poorly understood. This study was aimed to investigate the mechanisms mediating the toxic effects of MnCl(2), Mn(II) citrate, Mn(III) citrate, and Mn(III) pyrophosphate in primary cultures of neocortical (CTX) and cerebellar granular (CGC) neurons. Cell viability, mitochondrial function, and glutathione levels were evaluated after Mn exposure. CGC were significantly more susceptible to Mn-induced toxicity when compared with CTX. Moreover, undifferentiated CGC were more vulnerable to Mn toxicity than mature neurons. Mitochondrial dysfunction was observed after the exposure to all the tested Mn species. Ascorbate protected CGC against Mn-induced neurotoxicity, and this event seemed to be related to the dual role of ascorbate in neurons, acting as antioxidant and metabolic energetic supplier. CTX were protected from Mn-induced toxicity by ascorbate only when coincubated with lactate. These findings reinforce and extend the notion of the hazardous effects of Mn toward neuronal cells. In addition, the present results indicate that Mn-induced neurotoxicity is influenced by brain cell types, their origins, and developmental stages as well as by the chemical speciation of Mn, thus providing important information about Mn-induced developmental neurotoxicity and its risk assessment.

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species.

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.

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.

Localization of mitochondrial DNA base excision repair to an inner membrane-associated particulate fraction.

Mitochondrial DNA (mtDNA) contains high levels of oxidative damage relative to nuclear DNA. A full, functional DNA base excision repair (BER) pathway is present in mitochondria, to repair oxidative DNA lesions. However, little is known about the organization of this pathway within mitochondria. Here, we provide evidence that the mitochondrial BER proteins are not freely soluble, but strongly associated with an inner membrane-containing particulate fraction. Uracil DNA glycosylase, oxoguanine DNA glycosylase and DNA polymerase gamma activities all co-sedimented with this particulate fraction and were not dissociated from it by detergent (0.1% or 1.0% NP40) treatment. The particulate associations of these activities were not due to their binding mtDNA, which is itself associated with the inner membrane, as they also localized to the particulate fraction of mitochondria from 143B (TK-) rho(0) cells, which lack mtDNA. However, all of the BER activities were at least partially solubilized from the particulate fraction by treatment with 150-300 mM NaCl, suggesting that electrostatic interactions are involved in the association. The biological implications of the apparent immobilization of BER proteins are discussed.

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.

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA.

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK(-) osteosarcoma cells that had been depleted of mtDNA (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(0) cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(0) cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.

Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction.

Aging is strongly correlated with the accumulation of oxidative damage in DNA, particularly in mitochondria. Oxidative damage to both mitochondrial and nuclear DNA is repaired by the base excision repair (BER) pathway. The "mitochondrial theory of aging" suggests that aging results from declining mitochondrial function, due to high loads of damage and mutation in mitochondrial DNA (mtDNA). Restriction of caloric intake is the only intervention so far proven to slow the aging rate. However, the molecular mechanisms underlying such effects are still unclear. We used caloric-restricted (CR) mice to investigate whether lifespan extension is associated with changes in mitochondrial BER activities. Mice were divided into two groups, receiving 100% (PF) or 60% (CR) of normal caloric intake, a regime that extends mean lifespan by approximately 40% in CR mice. Mitochondria isolated from CR mice had slightly higher uracil (UDG) and oxoguanine DNA glycosylase (OGG1) activities but marginally lower abasic endonuclease and polymerase gamma gap-filling activities, although these differences were tissue-specific. Uracil-initiated BER synthesis incorporation activities were significantly lower in brain and kidney from CR mice but marginally enhanced in liver. However, nuclear repair synthesis activities were increased by CR, indicating differential regulation of BER in the two compartments. The results indicate that a general up-regulation of mitochondrial BER does not occur in CR.

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.

Age-associated increase in 8-oxo-deoxyguanosine glycosylase/AP lyase activity in rat mitochondria.

The mitochondrial theory of aging postulates that organisms age due to the accumulation of DNA damage and mutations in the multiple mitochondrial genomes, leading to mitochondrial dysfunction. Among the wide variety of DNA damage, 8-oxo-deoxyguanosine (8-oxo-dG) has received the most attention due to its mutagenicity and because of the possible correlation between its accumulation and pathological processes like cancer, degenerative diseases and aging. Although still controversial, many studies show that 8-oxo-dG accumulates with age in the mitochondrial (mt) DNA. However, little is known about the processing of this lesion and no study has yet examined whether mtDNA repair changes with age. Here, we report the first study on age-related changes in mtDNA repair, accomplished by assessing the cleavage activity of mitochondrial extracts towards an 8-oxo-dG-containing substrate. In this study, mitochondria obtained from rat heart and liver were used. We find that this enzymatic activity is higher in 12 and 23 month-old rats than in 6 month-old rats, in both liver and heart extracts. These mitochondrial extracts also cleave oligonucleotides containing a U:A mismatch, at the uracil position, reflecting the combined action of mitochondrial uracil DNA glycosylase (mtUDG) and mitochondrial apurinic/apyrimidinic (AP) endonucleases. The mtUDG activity did not change with age in liver mitochondria, but there was a small increase in activity from 6 to 23 months in rat heart extracts, after normalization to citrate synthase activity. Endonuclease G activity, measured by a plasmid relaxation assay, did not show any age-associated change in liver, but there was a significant decrease from 6 to 23 months in heart mitochondria. Our results suggest that the mitochondrial capacity to repair 8-oxo-dG, the main oxidative base damage suggested to accumulate with age in mtDNA, does not decrease, but rather increases with age. The specific increase in 8-oxo-dG endonuclease activity, rather than a general up-regulation of DNA repair in mitochondria, suggests an induction of the 8-oxo-dG-specific repair pathway with age.

Age-associated change in mitochondrial DNA damage.

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline. Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.

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.