Mitochondrial expression of the DNA repair enzyme OGG1 improves the prognosis of pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVES: 8-Hydroxydeoxyguanosine (8-OHdG) is an indicator of oxidative stress and causes transversion mutations and carcinogenesis. 8-OHdG is excision repaired by 8-OHdG DNA glycosylase 1 (OGG1), which is classified as nuclear and mitochondrial subtypes. We aimed to clarify the role of OGG1 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Ninety-two patients with PDAC who had undergone surgical resection at multiple institutions were immunohistochemically analyzed. The OGG1 and 8-OHdG expression levels were scored using the Germann Immunoreactive Score. The cutoff values of OGG1, as well as that of 8-OHdG, were determined. RESULTS: The low nuclear OGG1 expression group (n = 41) showed significantly higher carbohydrate antigen (CA)19-9 (p = 0.026), and higher s-pancreas antigen (SPAN)-1 (p = 0.017) than the high expression group (n = 51). Nuclear OGG1 expression has no effect on the prognosis. The low mitochondrial OGG1 expression group (n = 40) showed higher CA19-9 (p = 0.041), higher SPAN-1 (p = 0.032), and more histological perineural invasion (p = 0.037) than the high expression group (n = 52). The low mitochondrial OGG1 expression group had a significantly shorter recurrence-free survival (p = 0.0080) and overall survival (p = 0.0073) rates. The Cox proportional hazards model revealed that low mitochondrial OGG1 expression is an independent risk factor of the PDAC prognosis. OGG1 expression was negatively correlated with 8-OHdG expression (p = 0.0004), and high 8-OHdG expression shortened the recurrence-free survival of patients with PDAC. CONCLUSIONS: Low mitochondrial OGG1 expression might aggravate the PDAC prognosis.

Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability.

An important pathology in Parkinson's disease (PD) is the earlier and more severe degeneration of noradrenergic neurons in the locus coeruleus (LC) than dopaminergic neurons in the substantia nigra. However, the basis of such selective vulnerability to insults remains obscure. Using noradrenergic and dopaminergic cell lines, as well as primary neuronal cultures from rat LC and ventral mesencephalon (VM), the present study compared oxidative DNA damage response markers after exposure of these cells to hydrogen peroxide (H2O2). The results showed that H2O2 treatment resulted in more severe cell death in noradrenergic cell lines SK-N-BE(2)-M17 and PC12 than dopaminergic MN9D cells. Furthermore, there were higher levels of oxidative DNA damage response markers in noradrenergic cells and primary neuronal cultures from the LC than dopaminergic cells and primary cultures from the VM. It included increased tail moments and tail lengths in Comet assay, and increased protein levels of phosphor-p53 and γ-H2AX after treatments with H2O2. Consistent with these measurements, exposure of SK-N-BE(2)-M17 cells to H2O2 resulted in higher levels of reactive oxygen species (ROS). Further experiments showed that exposure of SK-N-BE(2)-M17 cells to H2O2 caused an increased level of noradrenergic transporter, reduced protein levels of copper transporter (Ctr1) and 8-oxoGua DNA glycosylase, as well as amplified levels of Cav1.2 and Cav1.3 expression. Taken together, these experiments indicated that noradrenergic neuronal cells seem to be more vulnerable to oxidative damage than dopaminergic neurons, which may be related to the intrinsic characteristics of noradrenergic neuronal cells.

Markers of HPA-axis activity and nucleic acid damage from oxidation after electroconvulsive stimulations in rats.

OBJECTIVE: Oxidative stress has been suggested to increase after electroconvulsive therapy (ECT), a treatment which continues to be the most effective for severe depression. Oxidative stress could potentially be mechanistically involved in both the therapeutic effects and side effects of ECT. METHODS: We measured sensitive markers of systemic and central nervous system (CNS) oxidative stress on DNA and RNA (urinary 8-oxodG/8-oxoGuo, cerebrospinal fluid 8-oxoGuo, and brain oxoguanine glycosylase mRNA expression) in male rats subjected to electroconvulsive stimulations (ECS), an animal model of ECT. Due to the previous observations that link hypothalamic-pituitary-adrenal (HPA)-axis activity and age to DNA/RNA damage from oxidation, groups of young and middle-aged male animals were included, and markers of HPA-axis activity were measured. RESULTS: ECS induced weight loss, increased corticosterone (only in middle-aged animals), and decreased cerebral glucocorticoid receptor mRNA expression, while largely leaving the markers of systemic and CNS DNA/RNA damage from oxidation unaltered. CONCLUSION: These results suggest that ECS is not associated with any lasting effects on oxidative stress on nucleic acids neither in young nor middle-aged rats.

Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.

Human Mpv17-like protein (M-LPH) has been suggested to participate in prevention of mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. To clarify the molecular mechanism of M-LPH function, we knocked out M-LPH in human hepatoma HepG2 using CRISPR-Cas9 technology. An increase in mtDNA damage in M-LPH-KO HepG2 cells was demonstrated by PCR-based quantitation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) measurement. Furthermore, confocal immunofluorescence analysis and Western blot analysis of mitochondrial extracts demonstrated that M-LPH-KO caused reductions in the protein levels of mitochondrial transcription factor A (TFAM), an essential factor for transcription and maintenance of mtDNA, and two DNA repair enzymes, 8-oxoguanine DNA glycosylase (OGG1) and DNA ligase 3 (LIG3), both involved in mitochondrial base excision repair (BER). Accordingly, it was suggested that the increase in mtDNA damage was due to a cumulative effect of mtDNA instability resulting from deficiencies of TFAM and diminished ability for BER arising from deficiencies in BER-related enzymes. These findings suggest that M-LPH could be involved in the maintenance of mtDNA, and therefore mitochondrial function, by protecting proteins essential for mtDNA stability and maintenance, in an integrated manner.

miR-200a Modulates the Expression of the DNA Repair Protein OGG1 Playing a Role in Aging of Primary Human Keratinocytes.

Oxidative DNA damage accumulation may induce cellular senescence. Notably, senescent cells accumulate in aged tissues and are present at the sites of age-related pathologies. Although the signaling of DNA strand breaks has been extensively studied, the role of oxidative base lesions has not fully investigated in primary human keratinocyte aging. In this study, we show that primary human keratinocytes from elderly donors are characterized by a significant accumulation of the oxidative base lesion 8-OH-dG, impairment of oxidative DNA repair, and increase of miR-200a levels. Notably, OGG1-2a, a critical enzyme for 8-OH-dG repair, is a direct target of miR-200a and its expression levels significantly decrease in aged keratinocytes. The 8-OH-dG accumulation displays a significant linear relationship with the aging biomarker p16 expression during keratinocyte senescence. Interestingly, we found that miR-200a overexpression down-modulates its putative target Bmi-1, a well-known p16 repressor, and up-regulates p16 itself. miR-200a overexpression also up-regulates the NLRP3 inflammasome and IL-1ß expression. Of note, primary keratinocytes from elderly donors are characterized by NRPL3 activation and IL-1ß secretion. These findings point to miR-200a as key player in primary human keratinocyte aging since it is able to reduce oxidative DNA repair activity and may induce several senescence features through p16 and IL-1ß up-regulation.

Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH.

DNA lesions caused by UV damage are thought to be repaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathway display a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led to the identification of the clinically approved anti-diabetic drug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expression of the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.

Enforced DNA repair enzymes rescue neurons from apoptosis induced by target deprivation and axotomy in mouse models of neurodegeneration.

It is unknown whether DNA damage accumulation is an upstream instigator or secondary effect of the cell death process in different populations of adult postmitotic neurons in the central nervous system. In two different mouse models of injury-induced neurodegeneration characterized by relatively synchronous accumulation of mitochondria, oxidative stress, and DNA damage prior to neuronal apoptosis, we enforced the expression of human 8-oxoguanine DNA glycosylase (hOGG1) and human apurinic-apyrimidinic endonuclease-1/Ref1 (hAPE) using recombinant adenoviruses (Ad). Thalamic lateral geniculate neurons and lumbar spinal cord motor neurons were transduced by Ad-hOGG1 and Ad-hAPE injections into the occipital cortex and skeletal muscle, respectively, prior to their target deprivation- and axotomy-induced retrograde apoptosis. Enforced expression of hOGG1 and hAPE in thalamus and spinal cord was confirmed by western blotting and immunohistochemistry. In injured populations of neurons in thalamus and spinal cord, a DNA damage response (DDR) was registered, as shown by localization of phospho-activated p53, Rad17, and replication protein A-32 immunoreactivities, and this DDR was attenuated more effectively by enforced hAPE expression than by hOGG1 expression. Enforced expression of hOGG1 and hAPE significantly protected thalamic neurons and motor neurons from retrograde apoptosis induced by target deprivation and axotomy. We conclude that a DDR response is engaged pre-apoptotically in different types of injured mature CNS neurons and that DNA repair enzymes can regulate the survival of retrogradely dying neurons, suggesting that DNA damage and activation of DDR are upstream mechanisms for this form of adult neurodegeneration in vivo, thus identifying DNA repair as a therapeutic target for neuroprotection.

Oxidized Guanine Base Lesions Function in 8-Oxoguanine DNA Glycosylase-1-mediated Epigenetic Regulation of Nuclear Factor κB-driven Gene Expression.

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.

Potential role of 8-oxoguanine DNA glycosylase 1 as a STAT1 coactivator in endotoxin-induced inflammatory response.

Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases.

Polymorphisms in NEIL-2, APE-1, CYP2E1 and MDM2 Genes are Independent Predictors of Gastric Cancer Risk in a Northern Jiangsu Population (China).

China is one of the countries with the highest incidence of gastric cancer, and accounts for over 40% of all new gastric cancer cases in the world. Genetic factors as well as environmental factors play a role in development of gastric cancer. To investigate the independent roles of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes (APE1 and NEIL2), carcinogen metabolism gene (CYP2E1) and tumor suppressor pathway gene (MDM2) for gastric cancer susceptibility in a Chinese population, we conducted a hospital based case-control study to evaluate the potential association between these polymorphisms and susceptibility to gastric cancer in a Northern Jiangsu population. We also associated the NEIL-2 mRNA expression with the studied NEIL2 SNP genotypes to assess whether the genotypes have influence on the NEIL2 mRNA (hence protein) expression. Five SNPs, APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920), were genotyped by TaqMan assays in 105 gastric cancer cases and 118 controls. Genotype frequency distribution showed that the APE 1 SNP (rs2275008), NEIL 2 SNP (rs804270), MDM2 SNP (rs2279744), and CYP 2E1 SNP (rs2031920) had more mutant alleles in gastric cancer cases than controls (76.19, 68.57, 54.29, and 43.81%, respectively), while CYP 2E1 SNP (rs2480256) had large percentage of both alleles (43.81%). Risk analysis revealed that there was increased risk for gastric cancer in subjects with mutant alleles in APE 1 (rs2275008: OR 5.49, 95% CI = 2.6-5.7, p <.0001), NEIL 2 (rs804270: OR 2.3, 95% CI = 1.22-4.3, p=0.01), MDM2 (rs2279744: OR 14.65, 95% CI = 5.63-8.15, p < .0001), and CYP 2E1 (rs2031920: OR 8.385, 95% CI = 3.2-5.3, p < .0001) SNPs. Moreover, the NEIL2 mRNA expression analysis showed that there was significant differential expression of NEIL2 mRNA among the randomly tested NEIL2 genotypes (p = 0.005), with low expression seen in variant genotypes than in other genotypes. In conclusion, variant alleles in the NEIL2 (rs804270), APE1 (rs2275008), CYP2E1 (rs2031920) and MDM2 (rs2279744) SNPs may independently influence susceptibility to gastric cancer in a Northern Jiangsu Chinese population. The genotypes may also independently influence their respective gene mRNA expression, as seen in our study, where there was differential expression of the NEIL2 mRNA among the genotypes, with low NEIL2 mRNA expression seen in the variant genotype.

Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo.

Oxidative DNA damage harbored by both spermatozoa and oocytes at the time of fertilization must be repaired prior to S-phase of the first mitotic division to reduce the risk of transversion mutations occurring in the zygote and subverting the normal patterns of cell differentiation and development. Of the characterised oxidative DNA lesions, 8-hydroxy-2'-deoxyguanosine (8OHdG) is particularly mutagenic. The current study reveals for the first time a marked acceleration of 8OHdG repair in the mouse oocyte/zygote by the base excision repair (BER) pathway following fertilization. Specifically, fertilization initiates post-translational modification to BER enzymes such as OGG1 and XRCC1, causing nuclear localisation and accelerated 8OHdG excision. Additionally, both the nuclear and mitochondrial genomes appear to benefit from increased protection against further 8OHdG formation by a fertilization-associated increase in glutathione peroxidase activity. The major limitation of the characterised 8OHdG repair system is the relatively low level of OGG1 expression in the oocyte, in contrast to the male germ line where it is the only constituent of the BER pathway. The male and female germ lines therefore collaborate in the repair of oxidative DNA damage, and oocytes are vulnerable to high levels of 8OHdG being carried into the zygote by the fertilizing spermatozoon.

Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

BACKGROUND: Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. OBJECTIVES: In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. RESULTS: Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. CONCLUSIONS: These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

The Phytoalexin Resveratrol Ameliorates Ochratoxin A Toxicity in Human Embryonic Kidney (HEK293) Cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50 = 1.5 µM (24 h) and 9.4 µM (48 h) determined using MTT assay] and 25 µM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P < 0.05), whereas OTA and OTA+resveratrol significantly decreased OGG1 expression (P < 0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTA+resveratrol (P < 0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTA+resveratrol yielded shorter comet tails (P < 0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTA+resveratrol significantly decreased mRNA expression of Nrf2 (P < 0.05). Luminometry analysis of GSH revealed an increase by OTA+resveratrol for 24 and 48 h (P < 0.05 and P < 0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P < 0.05) and OTA+resveratrol (P < 0.0011) and during 48-h exposure to resveratrol (P < 0.0005).

Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair.

RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

In vivo toxicity of cationic micelles and liposomes.

This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR: This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals.

Increased levels of oxidatively damaged DNA have been documented in studies of metal, metal oxide, carbon-based and ceramic engineered nanomaterials (ENMs). In particular, 8-oxo-7,8-dihydroguanine-2'-deoxyguanosine (8-oxodG) is widely assessed as a DNA nucleobase oxidation product, measured by chromatographic assays, antibody-based methods or the comet assay with DNA repair enzymes. However, spurious oxidation of DNA has been a problem in certain studies applying chromatographic assays, yielding high baseline levels of 8-oxodG. Antibody-based assays detect high 8-oxodG baseline levels, related to cross-reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations between exposure to ENMs and oxidized DNA in tissue than studies showing acceptable baseline levels (odds ratio = 12.1, 95% confidence interval: 1.2-124). Nevertheless, reliable studies indicate that intratracheal instillation of nanosized carbon black is associated with increased levels of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential.

Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8-hydroxydeoxyguanosine accumulation via activation of Akt and ERK1/2MAPK pathways.

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress.

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents ß-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.

Expression of 8-oxoguanine glycosylase in human fetal membranes.

PROBLEM: The most common DNA lesion generated by oxidative stress (OS) is 7, 8-dihydro-8-oxoguanine (8-oxoG) whose excision repair is performed by 8-oxoguanine glycosylase (OGG1). We investigated OGG1 expression changes in fetal membranes from spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) and its changes in vitro in normal fetal membranes exposed to OS inducer water-soluble cigarette smoke extract (CSE). METHOD OF STUDY: DNA damage was determined in amnion cells treated with CSE by comet and FLARE assays. OGG1 mRNA expression and localization in fetal membranes from clinical specimens and in normal term membranes exposed to CSE were examined by QRT-PCR and by immunohistochemistry. RESULTS: DNA strand and base damage was seen in amnion cells exposed to CSE. OGG1 expression was 2.5-fold higher in PTB samples compared with pPROM (P = 0.045). No significant difference was seen between term and pPROM or PTB and term. CSE treatment showed a nonsignificant decrease in OGG1. OGG1 was localized to both amnion and chorion with less intense staining in pPROM and CSE-treated membranes. CONCLUSION: Increased OS-induced DNA damage predominated by 8-oxoG is likely to persist in fetal cells due to reduced availability of base excision repair enzyme OGG1. This can likely lead to fetal cell senescence associated with some adverse pregnancy outcome.

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase γ was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage.