OGG1 Expression Research Articles

Oxidative stress in the thyroid – DNA damage and repair: Important mechanisms in the pathogenesis of thyroid tumours?

Oxidative stress due to an imbalance between pro-and antioxidative factors could be an important mechanism in the pathogenesis of thyroid diseases. We performed an IHC study to address the extent of 8-hydroxy-2'-deoxyguanine (8oxoG) formation, a key indicator of oxidative DNA base damage, in benign and malignant thyroid nodules. Besides we studied the enzyme expression of OGG-1, MYH and NTH responsible for repair of oxidative DNA damage. Ten toxic thyroid nodules (TTN), 14 cold benign thyroid nodules (CTN), their respective normal surrounding tissues (NT), 20 follicular (FTC), 18 papillary (PTC), 2 anaplastic (ATC), 5 medullary thyroid cancers (MTC) and 7 Graves' disease (GD) samples were investigated. Thyroid cancers showed a strong, morphology-specific subcellular 8oxoG staining pattern. In PTC, cytoplasmatic labelling was prevailing, suggesting vantaged mitochondrial DNA damage. In contrast, prominent nuclear staining was noticed in FTC. Comparison of differentially functioning benign thyroid nodules showed at least 2x increase in cytoplasmatic labelling in CTN compared to NT, while no difference was observed for TTN, their NT and GD. OGG-1, MYH and NTH mRNA expression was detected in all examined thyroid tissues with a significant downregulation of all 3 repair enzymes in FTC. In PTC we observed also a clear trend of downregulation but data failed to reach significance.

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.

Oxidative damage in nucleic acids and Parkinson's disease

Oxidative DNA lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial genomes during aging, and such accumulation can increase dramatically in patients with Parkinson's disease (PD). To counteract oxidative damage to nucleic acids, human and rodents are equipped with three distinct enzymes. One of these, MTH1, hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate, to their monophosphate forms. The other two enzymes are 8-oxoG DNA glycosylase encoded by the OGG1 gene and adenine/2-hydroxyadenine DNA glycosylase encoded by the MUTYH gene. We have shown a significant increase in 8-oxoG in mitochondrial DNA as well as an elevated expression of MTH1, OGG1, and MUTYH in nigrostriatal dopaminergic neurons of PD patients, suggesting that the buildup of these lesions may cause dopamine neuron loss. We established MTH1-null mice and found that MTH1-null fibroblasts were highly susceptible to cell death caused by H(2)O(2) characterized by pyknosis and electron-dense deposits in the mitochondria, and that this was accompanied by an ongoing accumulation of 8-oxoG in nuclear and mitochondrial DNA. We also showed that MTH1-null mice exhibited an increased accumulation of 8-oxoG in striatal mitochondrial DNA, followed by more extreme neuronal dysfunction after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration than that of wild-type mice. In conclusion, oxidative damage in nucleic acids is likely to be a major risk factor for Parkinson's disease, indicating that a solid understanding of the defense mechanisms involved will enable us to develop new strategies for protecting the brain against oxidative stress.

The capacity to remove 8-oxoG is enhanced in newborn neural stem/progenitor cells and decreases in juvenile mice and upon cell differentiation

In mammalian cells, 8-oxoguanine DNA glycosylase-1 (OGG1) is the main DNA glycosylase for the removal of 8-oxoguanine (8-oxoG). 8-oxoG, one of the most common products of the oxidative attack of DNA, is a premutagenic lesion that accumulates spontaneously at high frequencies in the genome. In this study, Ogg1 mRNA expression was detected throughout embryonic development in mice. In situ hybridization showed that in the neonatal brain, Ogg1 expression was detected in a distinct layer of cells in the medial wall of the lateral ventricle, which may correspond to ependymal cells, and in some scattered cells in the subventricular zone (SVZ), a brain region rich in neural stem/progenitor cells. Using neurospheres as a model for the study of neural stem/progenitor cells, we found that both the expression and activity of Ogg1 were high in neurospheres derived from newborn mice and decreased in adults and upon induction of cell differentiation. Furthermore, Ogg1 was shown to be the major DNA glycosylase initiating 8-oxoG repair in neurospheres. Our results strongly indicate that enhanced DNA repair capacity is an important mechanism by which neural stem/progenitor cells maintain their genome.

A novel R229Q OGG1 polymorphism results in a thermolabile enzyme that sensitizes KG-1 leukemia cells to DNA damaging agents

Background: Mutations and polymorphisms of OGG1, the major mammalian 8-oxoguanine repair activity, are associated with increased risk for several cancers. Decreased 8-oxoguanine repair capacity due to variant forms of the OGG1 gene is a common feature of numerous cancer cell lines. One such cell line, human KG-1 leukemia cells, has previously been demonstrated to be deficient in the excision of 8-oxoguanine from oxidatively damaged DNA. KG-1 cells have a homozygous R229Q amino acid substitution in OGG1 that has been presumed to alter the function of OGG1 and result in elevated levels of genomic 8-oxoG and hypersensitivity to 8-hydroxydeoxyguanosine nucleoside and ionizing radiation observed in KG-1 cells. Methods: We characterized the enzymatic activity of R229Q OGG1 and the effect of the enzyme on cell survival following treatment with DNA damaging agents. Results: R229Q OGG1 had activity similar to the wild-type enzyme, yet was easily heat inactivated at physiological temperature. R229Q OGG1 expressed in human cells had significantly lower activity than wild-type OGG1 and was also highly thermolabile. Expression of R229Q OGG1 sensitized KG-1 cells to killing by menadione and 8-hydroxydeoxyguanosine, but not ionizing radiation. Conclusions: These results suggest that decreased 8-oxoguanine repair in KG-1 is due to thermolability of R229Q OGG1 and that the enzyme variant increases cellular susceptibility to killing resulting from oxidative DNA damage. The R229Q OGG1 variant is a validated polymorphism prevalent in world populations and not an isolated mutation in KG-1 cells, thus the R229Q OGG1 allele may be a novel marker for cancer susceptibility.

Defects of Base Excision Repair Involving OGG1 Can Be Detected in a Subset of Patients with MDS and May Convey Propensity to Chromosomal Breaks.

Increased frequency of chromosomal lesions in MDS points towards defects in the DNA repair machinery as pathogenetic factors responsible for clonal evolution. Base excision repair (BER) is a possible mechanism of genomic instability. Upon genotoxic stress or due to defective function of the components of the BER, single-stranded breaks accumulate and are converted into double stranded breaks during mitosis. Oxidative damage may result in generation of 8-oxoguanine (OG), the most abundant DNA lesion. OG can be quantitated in hematopoietic cells using flow cytometry and we found significantly increased content of OG in patients with MDS (n=29, p=.0088). We stipulated that this observation may be a result of overwhelmed repair mechanism due to its insufficiency or inability to handle significantly increased oxidative stress. Here, we investigated this phenomenon in a subgroup of patients with increased genomic OG content (i.e. OG fluorescence >mean+2SD of controls). Remarkably, these patients were characterized by advanced stage of MDS (p=.0027), more profound cytopenias (neutropenia p=.001, thrombocytopenia p=.014) and propensity for AML transformation (p=.036). As OGG1 is the initial glycosylase that excises OG, we investigated the expression levels of this enzyme in highly purified CD34+ progenitor and stem cells in controls and patients with a high OG score; we found two expression patterns allowing for further sub-classification of affected patients. Low OGG1 (24/34) pointed towards a subset of patients with an inherent lesion that required further investigation. In contrast high OGG1 levels, found in 10/34 patients, were either consistent with an appropriate response to ongoing oxidative stress or the effect of a positive feedback due to low enzymatic activity. The SNP S326C of OGG1 has been associated with impaired function of the enzyme and compensatory upregulation of OGG1 transcription. This SNP is present in heterozygous form in controls at the frequency of 29%, but we found it in 8/10 patients with a high OGG1 expression, while 9/12 patients with wild-type had decreased expression (p=.01). In general, MDS patients (n=63) showed 6% homozygosity (vs. 0% in controls, p=.15) and 41% heterozygosity (vs 29% in controls, p=.24). Consequently, increased OG content could indicate a dysfunction of OGG1 resulting from the S326C SNP that cannot be adequately compensated. In fact, in 3/5 patients with OGG1 variant, an increased OG content and expression of OGG1 were found. Regardless of the activity of the OGG1, its upregulation could have various consequences to the downstream elements of the BER such as Polβ, which fills in the gaps left by the activity of endonucleases. Using Taqman RT-PCR performed on CD34+ cells; we tested Polβ expression in patients with upregulation of OGG1 and identified 2 groups of patients. The high Polβ group points toward overwhelming oxidative stress despite of adequate feedback response. In contrast, the low Polβ group could represent a subset of patients with defective upstream elements of the BER including endonuclease. This theory can be supported by elevated numbers of apurinic sites, detected by Elisa (0.54 +/− 0.70 in controls vs 1.5 +/− 2.1 in MDS AP sites/105bp). Taken together our results indicate that various lesions can contribute to the dysfunction of BER that occurs in MDS in the context of increased oxidative stress.

Increased mRNA expression levels of ERCC1, OGG1 and RAIin colorectal adenomas and carcinomas

BackgroundThe majority of colorectal cancer (CRC) cases develop through the adenoma-carcinoma pathway. If an increase in DNA repair expression is detected in both early adenomas and carcinomas it may indicate that low repair capacity in the normal mucosa is a risk factor for adenoma formation.MethodsWe have examined mRNA expression of two DNA repair genes, ERCC1 and OGG1 as well as the putative apoptosis controlling gene RAI, in normal tissues and lesions from 36 cases with adenomas (mild/moderat n = 21 and severe n = 15, dysplasia) and 9 with carcinomas.ResultsComparing expression levels of ERCC1, OGG1 and RAI between normal tissue and all lesions combined yielded higher expression levels in lesions, 3.3-fold higher (P = 0.005), 5.6-fold higher(P < 3·10-5) and 7.7-fold higher (P = 0.0005), respectively. The levels of ERCC1, OGG1 and RAI expressions when comparing lesions, did not differ between adenomas and CRC cases, P = 0.836, P = 0.341 and P = 0.909, respectively. When comparing expression levels in normal tissue, the levels for OGG1 and RAI from CRC cases were significantly lower compared to the cases with adenomas, P = 0.012 and P = 0.011, respectively.ConclusionOur results suggest that increased expression of defense genes is an early event in the progression of colorectal adenomas to carcinomas.

Chronic Arsenic Exposure and Oxidative Stress: OGG1 Expression and Arsenic Exposure, Nail Selenium, and Skin Hyperkeratosis in Inner Mongolia

Arsenic, a human carcinogen, is known to induce oxidative damage to DNA. In this study we investigated oxidative stress and As exposure by determining gene expression of OGG1, which codes for an enzyme, 8-oxoguanine DNA glycosylase, involved in removing 8-oxoguanine in As-exposed individuals. Bayingnormen (Ba Men) residents in Inner Mongolia are chronically exposed to As via drinking water. Water, toenail, and blood samples were collected from 299 Ba Men residents exposed to 0.34–826 μg/L As. RNA was isolated from blood, and mRNA levels of OGG1 were determined using real-time polymerase chain reaction. OGG1 expression levels were linked to As concentrations in drinking water and nails, selenium concentrations in nails, and skin hyperkeratosis. OGG1 expression was strongly associated with water As concentrations (p < 0.0001). Addition of the quadratic term significantly improved the fit compared with the linear model (p = 0.05). The maximal OGG1 response was at the water As concentration of 149 μg/L. OGG1 expression was also significantly associated with toenail As concentrations (p = 0.015) but inversely associated with nail Se concentrations (p = 0.0095). We found no significant differences in the As-induced OGG1 expression due to sex, smoking, or age even though the oldest group showed the strongest OGG1 response (p = 0.0001). OGG1 expression showed a dose-dependent increased risk of skin hyperkeratosis in males (trend analysis, p = 0.02), but the trend was not statistically significant in females. The results from this study provide a linkage between oxidative stress and As exposure in humans. OGG1 expression may be useful as a biomarker for assessing oxidative stress from As exposure.

Induction of 8-oxoguanine DNA Glycosylase 1 Gene Expression by HIV-1 Tat

In order to identify the cellular gene target for Tat, we have performed gene expression profile analysis and found that Tat upregulates the expression of 8-oxoguanine DNA glycosylase (OGG1) gene, encoding an enzyme responsible for repairing the oxidatively damaged guanine, 7,8-dihydro-8-oxoguanine (8-oxo-dG). We observed that Tat induces OGG1 gene expression by enhancing its promoter activity without changing the mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacts with AP-4 and removes AP-4 from OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8oxo-dG that causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked-down by small interfering RNA (siRNA), Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate a possibility that Tat may play a role in the maintenance of genetic integrity of the proviral and host cellular genomes by upregulating OGG1 as a feed-forward mechanism. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Ischemic Preconditioning in the Rat Brain Enhances the Repair of Endogenous Oxidative DNA Damage by Activating the Base-Excision Repair Pathway

The development of ischemic tolerance in the brain, whereby a brief period of sublethal 'preconditioning' ischemia attenuates injury from subsequent severe ischemia, may involve the activation of multiple intracellular signaling events that promote neuronal survival. In this study, the potential role of inducible DNA base-excision repair (BER), an endogenous adaptive response that prevents the detrimental effect of oxidative DNA damage, has been studied in the rat model of ischemic tolerance produced by three episodes of ischemic preconditioning (IP). This paradigm of IP, when applied 2 and 5 days before 2-h middle cerebral artery occlusion (MCAO), significantly decreased infarct volume in the frontal-parietal cortex 72 h later. Correlated with this protective effect, IP markedly attenuated the nuclear accumulations of several oxidative DNA lesions, including 8-oxodG, AP sites, and DNA strand breaks, after 2-h MCAO. Consequently, harmful DNA damage-responsive events, including NAD depletion and p53 activation, were reduced during postischemic reperfusion in preconditioned brains. The mechanism underlying the decreased DNA damage in preconditioned brain was then investigated by measuring BER activities in nuclear extracts. Beta-polymerase-mediated BER activity was markedly increased after IP, and this activation occurred before (24 h) and during the course of ischemic tolerance (48 to 72 h). In similar patterns, the activities for AP site and 8-oxodG incisions were also upregulated after IP. The upregulation of BER activities after IP was likely because of increased expression of repair enzymes beta-polymerase, AP endonuclease, and OGG1. These results suggest that the activation of the BER pathway may contribute to IP-induced neuroprotection by enhancing the repair of endogenous oxidative DNA damage after ischemic injury.

Induction of OGG1 Gene Expression by HIV-1 Tat

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by up-regulating OGG1 as a feed-forward mechanism.

Allelic loss of the DNA repair gene OGG1 against oxidative damage in esophageal squamous cell carcinoma

Esophageal tissue often undergoes oxidative damage from exposure to cigarettes and alcohol. The OGG1 gene plays a major role in maintaining genetic integrity by removing 8-oxoguanine (8-oxoG) in cellular DNA by way of the base excision repair pathway. The OGG1 gene is located at 3p26.2 of the human chromosome. Although deletion in the 3p region is frequently found in cases of esophageal cancer, few reports have focused on the allelic loss of the OGG1 gene. Using 24 samples of surgically-resected esophageal cancer tissue, we assessed the allelic loss of the OGG1 gene with a set of three microsatellite markers that flank the OGG1 gene. We compared the loss of homozygosity status for the OGG1 gene with clinicopathologic factors, as well as OGG1 expression and accumulation of 8-oxoG, which was estimated in an immunohistochemical study. Of 24 cases, 20 were suitable for determining OGG1 allelic loss. Allelic loss of OGG1 was observed in 6 of 20 (30%) cases. An inverse correlation between OGG1 allelic loss and OGG1 expression was weakly observed (p=0.051). In contrast, OGG1 allelic loss showed a positive correlation with 8-oxoG accumulation, although the correlation was not statistically significant (0.095). OGG1 allelic loss was observed in 30% of patients in our study, a lower frequency than that reported in other malignancies, and correlated with reduced OGG1 expression and 8-oxoG accumulation. These findings suggest that attenuated OGG1 expression contributes to 8-oxoG accumulation under oxidative stress, resulting in cancer development in the esophagus. Thus, OGG1 allelic loss is related to carcinogenesis by oxidative stress rather than cancer progression in esophageal cancer.

P21Cip1/WAF1/Sdi1Does Not Affect Expression of Base Excision DNA Repair Enzymes During Chronic Oxidative Stress

Exposure to chronic oxidative stress during elevated oxygen (hyperoxia) damages DNA and inhibits cell proliferation in G(1) through induction of the cyclin-dependent kinase inhibitor p21. Cells that fail to express p21 growth-arrest in S phase. The observation that growth arrest in G(1) is associated with reduced DNA damage and enhanced survival suggests that p21 may affect expression of base excision repair (BER) enzymes used to repair oxidized DNA. This hypothesis was tested in p21 wild-type and p21-deficient mice and human lung adenocarcinoma H1299 cells with tetracycline-on regulated expression of p21. The mRNA levels of Ogg1, Tdg, Udg, Mpg, Nth1, and Mgmt remained constant during 3 days of hyperoxia. The expression of Ogg1, Nth1, and APE protein also remained unchanged. Although hyperoxia increased p21, its absence did not significantly affect expression of these repair enzymes. These findings reveal that hyperoxia induces p21 without significantly altering BER enzyme expression. This suggests that p21 may protect oxidized cells by affecting the activity of BER enzymes and/or through other mechanisms, such as apoptosis.

DNA damage in lung after oral exposure to diesel exhaust particles in Big Blue ® rats

Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts. Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue ® rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks (comet assay) and bulky DNA adducts ( 32 P post labeling) was observed. The level of DNA strand breaks increased significantly at all dose levels while the level of DNA adducts increased significantly only at the intermediate dose levels. Similarly, the number of oxidized DNA bases measured as endonuclease III and fapyguanine glycosylase (FPG) sensitive sites increased at the intermediate dose levels. The induction of DNA damage by DEP exposure did not increase the expression of the repair genes OGG1 and ERCC1 at the mRNA level. The present study indicates that the lung is a target organ for primary DNA damage following oral exposure to DEP. DNA damage was induced following exposure to relatively low levels of DEP, but under the conditions used in the present experiment DNA damage did not result in an increased mutation rate.

Biological Significance of the Defense Mechanisms against Oxidative Damage in Nucleic Acids Caused by Reactive Oxygen Species: From Mitochondria to Nuclei

Abstract: In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8‐oxo‐dGTP, 8‐oxo‐dATP, and 2‐hydroxy (OH)‐dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1‐deficient mice and an alteration of MTH1 expression along with the accumulation of 8‐oxo‐dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8‐oxoG DNA glycosylase (OGG1), 2‐OH‐A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH‐deficient mice. The increased occurrence of lung tumor in OGG1‐deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

Transcription Factors NF-YA Regulate the Induction of Human OGG1 Following DNA-alkylating Agent Methylmethane Sulfonate (MMS) Treatment

A human 8-oxoguanine-DNA glycosylase (hOGG1) is the main enzyme that repairs 8-oxoG, which is a critical mutagenic lesion. There is a great deal of interest in the up- or down-regulation of OGG1 expression after DNA damage. In this study, we investigated the effect of a DNA-alkylating agent, methylmethane sulfonate (MMS), on hOGG1 expression level and found that MMS treatment resulted in an increase in the functional hOGG1 expression in HCT116 cells. A region between -121 and -61 of the hOGG1 promoter was found to be crucial for this induction by MMS. Site-directed mutations of the two inverted CCAAT motifs substantially abrogated the induction of the hOGG1 promoter as a result of MMS treatment. In addition, the NF-YA protein (binding to the inverted CCAAT box) was induced after exposing cells to MMS. Moreover, gel shift and supershift analyses with the nuclear extracts prepared from HCT116 cells identified NF-YA as the transcription factor interacting with the inverted CCAAT box. Mutations of the inverted CCAAT box either prevented the binding of this factor or abolished its activation as a result of MMS treatment. Finally, this study showed that hOGG1-expressing HCT116 cells exhibited increased hOGG1 repair activity and resistance to MMS. Overall, these results demonstrate that MMS can up-regulate hOGG1 expression through the induction of the transcription factor, NF-YA, and increased transcription level of the hOGG1 gene correlates with an increase in enzyme activity providing functional protection from MMS.

Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei.

In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

X-ray-induced Oxidative Stress: DNA Damage and Gene Expression of HO-1 , ERCC1 and OGG1 in Mouse Lung

Effects of X-ray induced oxidative stress in mouse lungs were studied in terms of DNA damage and expression of antioxidant defense and DNA repair genes. Lung samples were collected immediately after, and 3, 6, and 22 h after irradiation with 1, 3, 10 or 30 Gy X-rays of the thorax. The levels of strand breaks (SB), formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII) sensitive sites, detected by the comet assay, were increased dose-dependently immediately after irradiation, whereas 8-oxo-7,8-dihydro-2'-deoxyguanosine analyzed by HPLC-EC was unaltered, possibly due to a relatively high background level (2.5/10(6) dG in control tissue). Complete repair of SB was observed 3 h after irradiation, whereas the period required for repair of ENDOIII and FPG sensitive sites was longer. Determined by RT-PCR, the mRNA expression of heme oxygenase-1 (HO-1) was increased 40-fold 6 h after irradiation, whereas the expression of 8-oxoguanine glycosylase (OGG1) and ERCC1 were increased 2.5-fold 6 h after exposure, with saturation at the lowest dose. In conclusion, this study shows the feasibility of partial-body X-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, and expression of relevant DNA repair and antioxidant defense genes.

Oxidative DNA damage and defence gene expression in the mouse lung after short-term exposure to diesel exhaust particles by inhalation.

Exposure to diesel exhaust particles (DEP) is suspected to contribute to lung cancer and cardiopulmonary diseases. In recent years generation of reactive oxygen species capable of inducing cellular oxidative stress has been in focus as one of the underlying mechanisms behind the genotoxic effects of particles. However, the role of the antioxidative defence system still needs to be clarified, especially in relation to low-dose DEP exposures. The aim of this study was to characterize the effects of short-term exposure to DEP in terms of DNA damage and expression of key response genes towards oxidative stress in lungs of mice. Mice were exposed by inhalation to 20 or 80 mg/m3 DEP inhaled as either a single dose, or four lower doses (5 and 20 mg/m3) inhaled on four consecutive days. Our results indicate that HO-1 mRNA expression in lung tissue was up-regulated after both types of DEP exposures, whereas OGG1 expression was only up-regulated after repeated exposures. The level of oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased in the lung tissue after a single exposure, whereas increased levels of DNA strand breaks was observed in bronchoalveolar lavage cells after repeated DEP exposures. The levels of 8-oxodG and OGG1 mRNA in lung tissue were mirror images. This suggests that after repeated exposures, up-regulation of DNA repair counteracts an increased rate of 8-oxodG formation leaving the steady state level of 8-oxodG in DNA unchanged. In conclusion, this study indicates that a single high dose of DEP generates 8-oxodG in lung tissue, whereas the same dose inhaled as four low-exposures may up-regulate the antioxidative defence system and protect against generation of 8-oxodG.

DNA adduct formation and oxidative stress in colon and liver of Big Blue(R) rats after dietary exposure to diesel particles

Exposure to diesel exhaust particles (DEP) via the gastrointestinal route may impose risk of cancer in the colon and liver. We investigated the effects of DEP given in the diet to Big Blue rats by quantifying a panel of markers of DNA damage and repair, mutation, oxidative damage to proteins and lipids, and antioxidative defence mechanisms in colon mucosa cells, liver tissue and the blood compartment. Seven groups of rats were fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. DEP induced a significant increase in DNA strand breaks in colon and liver. There was no effect on oxidative DNA damage (8-oxodG) in colon or liver DNA or in the urine. However, the mRNA expression of OGG1, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by 32P-post-labelling were elevated in colon and liver, and the expression of ERCC1 gene was affected in liver, but not in colon. In addition to these effects, DEP exposure induced apoptosis in liver. There was no significant change in mutation frequency in colon or liver. The levels of oxidative protein modifications (oxidized arginine and proline residues) were increased in liver accompanied by enhanced vitamin C levels. In plasma, we found no significant effects on oxidative damage to proteins and lipids, antioxidant enzymes or vitamin C levels. Our data indicate that gastrointestinal exposure to DEP induces DNA adducts and oxidative stress resulting in DNA strand breaks, enhanced repair capacity of oxidative base damage, apoptosis and protein oxidation in colon mucosa cells and liver.