8-hydroxyguanine and Lung-Neoplasms

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

Topics: Aging; Alveolar Epithelial Cells; Animals; Apoptosis; Disease Models, Animal; DNA Damage; DNA Glycosylases; DNA Repair; DNA, Mitochondrial; Guanine; Humans; Lung Neoplasms; Mitochondria; Oxidative Stress; Pulmonary Fibrosis; Reactive Oxygen Species; Sirtuin 3

DNA repair has a major role in suppressing the rate of accumulation of mutations. Therefore, variations in DNA repair are likely to play an important role in determining cancer risk. While there is compelling evidence that defects in DNA repair cause high predisposition to several hereditary cancers, there is a paucity of data on the role of DNA repair in sporadic cancers. We present our approach of using functional DNA repair tests, rather than gene polymorphism, to study the potential of DNA repair enzymes to serve as biomarkers for lung cancer risk. We have previously developed a functional DNA repair blood test for the enzymatic repair of the oxidative DNA lesion 8-oxoguanine, and found that reduced OGG activity is a risk factor in non-small cell lung cancer. Moreover the combination of smoking and low OGG activity was associated with a greatly increased lung cancer risk (Paz-Elizur et al, JNCI 95 (2003) 1312-1319). The use of OGG activity as a potential biomarker for lung cancer risk is validated in collaboration with the M. D. Anderson Cancer Center, under the sponsorship of the Associate Members Program of the Early Detection Research Network (EDRN, NCI, NIH).

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease Susceptibility; DNA Damage; DNA Glycosylases; DNA Repair; Guanine; Humans; Lung Neoplasms; Oxidative Stress; Risk Assessment

To assess oxidative damage to DNA during lung cancer (LC) treatments.. Urinary levels of 8-oxoguanine (8-oxoGua) and levels of 8-oxo-2'-deoxyguanosine (8-oxodG) from urine and whole blood were determined in 36 non-cancer controls and 65 LC patients before any treatments. Samples were also obtained of LC patients during and after radiotherapy (RT, n=33) and chemotherapy (CT, n=16).. Stage IV LC patients had higher urinary 8-oxoGua and 8-oxodG levels than patients with stage I-III disease (p=0.044 and p=0.034, respectively). Urinary 8-oxodG levels increased during the first week of RT (p<0.001). Nuclear 8-oxodG increased during RT and 3 months after start of RT. Nuclear 8-oxodG levels also rose between the first two CT cycles (p=0.043), and urinary 8-oxodG levels during the sixth CT cycle (p=0.009).. Urinary DNA damage biomarker levels may be associated with LC stage. Both RT and CT increase the parameters of DNA oxidation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Aged, 80 and over; Biomarkers, Tumor; Demography; Deoxyguanosine; DNA Damage; Female; Guanine; Humans; Lung Neoplasms; Male; Middle Aged; Oxidative Stress; Survival Analysis; Treatment Outcome

Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means of the comet assay. We collected blood samples from 1,019 healthy subjects and genotyped for the OGG1 Ser326Cys polymorphism. We found 49 subjects homozygous for the variant genotype (Cys/Cys) and selected same numbers of age-matched subjects with the heterozygous (Ser/Cys) and homozygous wild-type genotype (Ser/Ser). Carriers of the Cys/Cys genotype had higher levels of formamidopyrimidine DNA glycosylase (FPG) sensitive sites in MNBC (0.31 ± 0.03 lesions/10(6)bp) compared to Ser/Ser (0.19 ± 0.02 lesions/10(6)bp, P<0.01). The level of hOGG1 sensitive sites in MNBC from the Ser326Cys carriers (0.19 ± 0.16 lesions/10(6) bp) was also higher compared to the Ser/Ser genotype (0.11 ± 0.09 lesions/10(6) bp, P<0.05). Still, there was no genotype-related difference in DNA repair incision activity of MNBC extracts on nucleoids with oxidatively damaged DNA induced by Ro19-8022/white light (P=0.20). In addition, there were no differences in the expression of OGG1 (P=0.69), ERCC1 (P=0.62), MUTYH (P=0.85), NEIL1 (P=0.17) or NUDT1 (P=0.48) in whole blood. Our results indicate that the OGG1 Ser326Cys polymorphism has limited influence on the DNA repair incisions by extracts of MNBC, whereas the apparent increased risk of cancer in subjects with the Cys/Cys genotype may be because of higher levels of oxidatively damaged DNA.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Case-Control Studies; Comet Assay; Denmark; DNA Damage; DNA Glycosylases; DNA Repair; Female; Genetic Predisposition to Disease; Genotype; Guanine; Homozygote; Humans; Leukocytes, Mononuclear; Lung Neoplasms; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress; Polymorphism, Single Nucleotide

Oxidative damage to guanine (8-oxoGua) is one of the most abundant lesions induced by oxidative stress and documented mutagenic. 8-Oxoguanine DNA glycosylase 1 (OGG1) removes 8-oxoGua from DNA by excision. The urinary excretion of 8-oxoGua is a biomarker of exposure, reflecting the rate of damage in the steady state. The aim of this study was to investigate urinary 8-oxoGua as a risk factor for lung cancer. In a nested case-cohort design we examined associations between urinary excretion of 8-oxoGua and risk of lung cancer as well as potential interaction with the OGG1 Ser326Cys polymorphism in a population-based cohort of 25,717 men and 27,972 women aged 50-64 years with 3-7 years follow-up. We included 260 cases with lung cancer and a subcohort of 263 individuals matched on sex, age, and smoking duration for comparison. Urine collected at entry was analysed for 8-oxoGua by HPLC with electrochemical detection. There was no significant effect of smoking or OGG1 genotype on the excretion of 8-oxoGua. Overall the incidence rate ratio (IRR) (95% confidence interval) of lung cancer was 1.06 (0.97-1.15) per doubling of 8-oxoGua excretion. The association between lung cancer risk and 8-oxoGua excretion was significant among men [IRR: 1.17 (1.03-1.31)], never-smokers [IRR: 9.94 (1.04-94.7)], and former smokers [IRR: 1.19 (1.07-1.33)]. There was no significant interaction with the OGG1 genotype, although the IRR was 1.14 (0.98-1.34) among subjects homozygous for Cys326. The association between urinary 8-oxoGua excretion and lung cancer risk among former and never-smokers suggests that oxidative stress with damage to DNA is important in this group.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Case-Control Studies; Chromatography, High Pressure Liquid; Denmark; DNA Glycosylases; Female; Follow-Up Studies; Genotype; Guanine; Homozygote; Humans; Incidence; Lung Neoplasms; Male; Middle Aged; Oxidative Stress; Polymorphism, Genetic; Population Surveillance; Prospective Studies; Risk Factors; Smoking

This study was aimed at defining the reference ranges for biomarkers of oxidized guanine in (2'-deoxy)ribonucleotides and nucleic acids from a large Italian sample. We recruited 300 healthy subjects (150 males; mean age 44.1±13.6years; 26% smokers) without any known exposure to occupational oxidizing agents. They were asked to provide a spot urine sample, on which the following markers were determined by liquid chromatography-tandem mass spectrometry: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), 8-oxo-7,8-dihydroguanine (8-oxoGua), and cotinine. The reference ranges, estimated as the 5th-95th percentiles of creatinine-normalized values (pmol/μmol(creat)) were 0.7-4.2, 0.9-4.7, and 5.6-120.7 for 8-oxodGuo, 8-oxoGuo, and 8-oxoGua, respectively. Oxidation biomarkers were correlated with one another (p<0.005) and with urinary creatinine (p<0.0001). Males excreted significantly higher concentrations of 8-oxoGua than females (p<0.0001). 8-OxoGua and 8-oxoGuo showed a positive association with age (p<0.001), also after stratification by gender. Multiple linear regression models including urinary creatinine concentration, age, and smoking habit as independent variables showed a significant effect of age, but not of smoking, on the levels of 8-oxoGuo in males (p<0.0001) and of both 8-oxoGuo and 8-oxoGua in females (p<0.0001). A preliminary assessment in a small group (n=25) of patients affected by advanced non-small-cell lung cancer and receiving platinum-based chemotherapy showed significantly higher values of both 8-oxoGuo and 8-oxodGuo (p<0.0001 for both) compared to the referent population.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers; Carcinoma, Non-Small-Cell Lung; Chromatography, Liquid; Cotinine; Deoxyguanosine; Female; Guanine; Guanosine; Humans; Lung Neoplasms; Male; Middle Aged; Nucleic Acids; Oxidation-Reduction; Oxygen; Reference Values; Smoking; Tandem Mass Spectrometry; Young Adult

Decreased repair of oxidative DNA damage is a risk factor for developing certain human malignancies. We have previously found that the capacity of 8-oxo-7,8-dihydroguanine repair was lower in leukocytes of NSCLC patients than in controls. To explain these observations, we searched for mutations and polymorphisms in the OGG1 gene among 88 NSCLC patients and 79 controls. One patient exhibited a heterozygous mutation in exon 1, which resulted in Arg46Gln substitution. Normal lung and tumor tissue carrying this mutation showed markedly lower 8-oxoG incision activity than the mean for all patients. The predominant polymorphism of OGG1 was Ser326Cys. A significant difference was observed in the frequencies of the OGG1 variants between populations of NSCLC patients and controls. The frequency of the Cys326 allele and the number of Cys326Cys homozygotes was higher among patients than controls. In individuals with either Ser326Cys or Cys326Cys genotype 8-oxoG incision rate was lower than in those with both Ser326 alleles, either in lung or leukocytes. Moreover, 8-oxodG level was higher in lung tissue and leukocytes of patients carrying two Cys326 alleles and in leukocytes of patients with the Ser326Cys genotype. We also screened for polymorphisms of the XRCC1 gene. Only heterozygotes of the XRCC1 variants Arg194Trp, Arg280His and Arg399Gln were found among patients and controls, with the frequency of Arg280His being significantly higher among patients. NSCLC patients with Arg280His or Arg399Gln polymorphism revealed lower 8-oxoG incision activity in their lung tissues, but not in leukocytes. We can conclude that the OGG1 Ser326Cys polymorphisms may have an impact on the efficiency of 8-oxoG incision in humans and the XRCC1 His280 and Gln399 may influence the OGG1 activity in tissues exposed to chronic oxidative/inflammatory stress. Higher frequency of the OGG1 Cys326 allele among NSCLC patients may partially explain the impairment of the 8-oxoG repair observed in their leukocytes.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Deoxyguanosine; DNA Repair; DNA-Binding Proteins; Female; Guanine; Humans; Leukocytes; Lung; Lung Neoplasms; Male; Middle Aged; Polymorphism, Genetic; X-ray Repair Cross Complementing Protein 1

To study the expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in non-small cell lung cancer (NSCLC), and the interaction between hnRNP A2/B1 protein and mRNA of DNA repair enzymes O(6)-methylguanine DNA-methyltransferase (MGMT), 8-oxoguanine DNA glycosylase (OGG1), redox factor 1(Ref-1), DNA-dependent protein kinase (including DNA-PKcs and ku).. The expression and distribution of hnRNP A2/B1 were detected by immunohistochemistry and Western blot on 50 NSCLC samples from patients who underwent resection in Zhongshan Hospital. The hnRNP A2/B1 mRNA expression was tested by real-time PCR. Co-immunoprecipitation (co-IP) combined RT-PCR was used to investigate whether hnRNP A2/B1 could be bound with the mRNA of the above mentioned 5 DNA repair enzymes in human lung cancer cell line (HTB-182). Then immunohistochemistry and real-time PCR were used to detect the expression of MGMT in the same group of patients.. HnRNP A2/B1 protein and mRNA expressions were increased in the NSCLC tissues than that in the corresponding normal lung tissues. HnRNP A2/B1 was expressed predominantly in the nuclei of tumor cells. The positive rate and immunohistochemistry score of hnRNP A2/B1 in tumor tissue were significantly higher than that in normal tissue (P < 0.01). In stage III-IV NSCLC, hnRNP A2/B1 expression was higher than that in stage I-II. There was no significant differences of hnRNP A2/B1 expression among patients of different age, sex, histological type, and smoking history. The results of co-IP combined RT-PCR suggested that hnRNP A2/B1 is bound with MGMT mRNA, and MGMT expression is decreased in tumor tissue of NSCLC.. The results of this study show that hnRNP A2/B1 protein and mRNA are highly expressed in NSCLC, and hnRNP A2/B1 is bound with MGMT mRNA, which indicate that it might be one of the mechanisms of hnRNP A2/B1 participating in the pathogenesis of NSCLC.

Topics: Blotting, Western; Carcinoma, Non-Small-Cell Lung; DNA-Activated Protein Kinase; Guanine; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Immunohistochemistry; Immunoprecipitation; Lung; Lung Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

DNA repair capacity is an important determinant of susceptibility to cancer. The hOGG1 enzyme is crucial for repairing the 8-oxoguanine lesion that occurs either as a byproduct of oxidative metabolism or as a result of exogenous sources such as exposure to cigarette smoke. It has been previously reported that smokers with low hOGG1 activity had significantly higher risk of developing lung cancer as compared to smokers with high hOGG1 activity.. In the current study we elucidate the association between plasma levels of 8-OHdG and the OGG1 repair capacity. We used the commercially available 8-OHdG ELISA (enzyme-linked immunosorbent assay), the Comet assay/FLARE hOGG1 (Fragment Length Analysis by Repair Enzymes) assay for quantification of the levels of 8-OHdG and measured the constitutive, induced and unrepaired residual damage, respectively. We compared the DNA repair capacity in peripheral blood lymphocytes following H2O2 exposure in 30 lung cancer patients, 30 non-, 30 former and 30 current smoker controls matched by age and gender.. Our results show that lung cancer cases and current smoker controls have similar levels of 8-OHdG lesions that are significantly higher compared to the non-smokers controls. However, lung cancer cases showed significantly poorer repair capacity compared to all controls tested, including the current smokers controls. After adjustment for age, gender and family history of smoking-related cancer using linear regression, we observed a 5-fold increase in risk of lung cancer associated with high levels of residual damage/reduced repair capacity. Reduced OGG1 activity could be expected to be a risk factor in other smoking-related cancers.. Our study shows that the Comet/FLARE assay is a relatively rapid and useful method for determination of DNA repair capacity. Using this assay we could identify individuals with high levels of residual damage and hence poor repair capacity who would be good candidates for intensive follow-up and screening.

Topics: Case-Control Studies; Comet Assay; DNA Damage; DNA Glycosylases; DNA Repair; Enzyme-Linked Immunosorbent Assay; Female; Guanine; Humans; Hydrogen Peroxide; Lung Neoplasms; Lymphocytes; Male; Middle Aged; Oxidants; Risk Factors; Smoking

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

Topics: Animals; Base Sequence; Brain; Deoxyribonuclease (Pyrimidine Dimer); DNA Damage; DNA Glycosylases; DNA Mutational Analysis; Female; Gas Chromatography-Mass Spectrometry; Gene Deletion; Genes, ras; Guanine; Kidney; Liver; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Mutation; Oxidation-Reduction; Pyrimidines

The potential roles of peroxiredoxin (Prx) I in carcinogenesis and treatment have been explored. Our previous study revealed differences between A549 (functional p53) and H1299 (null p53) Prx I antisense transfectants. The discrepancy might have resulted from the p53 status. In this study, we further investigated the role of Prx I and p53 on lung cancer growth and the response to treatment in vitro and in vivo.. We established stable A549 and H1299 transfectants with Prx I antisense and p53, respectively. We then examined their characteristics in vitro and used nude mice xenografts of these cell lines to compare their capacity for tumor invasion and spontaneous metastasis and their sensitivity to radiotherapy.. Increased reactive oxygen species caused by lower Prx I activity induced p53 expression. In lethal stress, the augmentation of reactive oxygen species was partially reversed by blocking p53 in A549 with Prx I antisense. We demonstrated the potential contribution of p53-dependent mechanisms to inhibit lung tumor growth and increase radiosensitization using H1299 transfected with p53 in vitro and in vivo. An increased p53 level attenuated the capacity of the cells for metastasis by decreasing vascular endothelial growth factor and induced radiosensitization by increased apoptosis and cell senescence and by regulating intracellular reactive oxygen species.. These results suggest that p53 status has an important role in the tumor-inhibiting and radiosensitizing effects of decreasing Prx I. Both Prx I and p53 may be powerful prognosticators for lung cancer.

Topics: Animals; Apoptosis; beta-Galactosidase; Cell Line, Tumor; Cell Proliferation; DNA Damage; Genes, p53; Guanine; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Peroxidases; Peroxiredoxins; Radiation Tolerance; Reactive Oxygen Species; Transfection; Tumor Suppressor Protein p53

In humans, the oxidatively induced DNA lesion 8-hydroxyguanine (8-oxoG) is removed from DNA by hOgg1, a DNA glycosylase/AP lyase that specifically incises 8-oxoG opposite cytosine. We analysed the expression of hOGG1 mRNA in 18 lung cancer and three normal cell lines. Although hOGG1 was overexpressed in most cell lines, 2/18 (11.1%) showed a lower hOGG1 mRNA and protein expression (approximately 80% decrease) relative to normal cell lines. Liquid chromatography/mass spectrometry analysis showed increased levels of 8-oxoG in the two cell lines with the lowest hOGG1 mRNA expression. We examined the ability of nuclear and mitochondrial extracts to incise 8-oxoG lesion in cell lines H1650 and H226 expressing lower hOGG1 mRNA and H1915 and H1975 with higher than normal hOGG1 mRNA expression. Both nuclear and mitochondrial extracts from H1915 and H1975 cells were proficient in 8-oxoG removal. However, both cell lines with the lowest hOGG1 mRNA expression exhibited a severe reduction in 8-oxoG incision in both nuclear and mitochondrial extracts. Under-expression of hOGG1 mRNA and hOgg1 protein was associated with a decrease in mitochondrial DNA repair in response to oxidative damaging agents. These results provide evidence for defective incision of 8-oxoG in both nuclear and mitochondria of H1650 and H226 lung cancer cell lines. These results may implicate 8-oxoG repair defects in certain lung cancers.

Topics: Cell Nucleus; Chromatography, Liquid; DNA Damage; DNA Glycosylases; DNA Repair; DNA, Mitochondrial; Gene Expression Regulation, Neoplastic; Guanine; Humans; Lung Neoplasms; Mass Spectrometry; Mitochondria; Oxidants; Oxidative Stress; Reference Values; Sequence Analysis; Tumor Cells, Cultured

The effect of hyperoxia on levels of DNA damage and global DNA methylation was examined in lung epithelial-like A549 cells. DNA damage was assessed by the single-cell gel electrophoresis (comet assay) and DNA methylation status by the cytosine extension assays. Cells exposed to ionizing radiation (0, 1, 2, 4, or 8 Gy) showed increasing rates of percentage of DNA in the tail and tail length with increasing radiation dose. When cells were exposed to room air (normoxia) for 1 day and 95% O2 (hyperoxia) for 1, 2, 3, 4, and 5 days, data indicated that hyperoxia caused time-dependent increases in levels of (a) single strand breaks, (b) double strand breaks, and (c) 8-oxoguanine. Decreased DNA methylation also was observed at day 5 of hyperoxic exposure, suggesting that hyperoxia-induced DNA damage can influence patterns of DNA methylation in a lung-derived cell line.

Topics: Cell Line, Tumor; Comet Assay; DNA; DNA Damage; DNA Methylation; DNA Repair; DNA, Single-Stranded; Epithelial Cells; Guanine; Humans; Hyperoxia; Lung Neoplasms; Oxygen; Time Factors

OGG1 protein has an ability to suppress mutagenesis induced by 8-hydroxyguanine (8OHG), an oxidatively damaged promutagenic base. Here, the mutation suppressive ability was compared between two common polymorphic OGG1 proteins, OGG1-Ser326 and OGG1-Cys326, using a supF forward mutation assay employing an 8OHG-containing plasmid. Polymorphic OGG1 proteins were exogenously expressed by adenoviral transduction in H1299 human lung cancer cells, in which endogenous OGG1 protein was undetectable by western blot analysis. Mutations by 8OHG were more efficiently suppressed in OGG1-Ser326 transduced cells than OGG1-Cys326 transduced cells. The results indicated that OGG1-Cys326 has a lower ability to prevent mutagenesis by 8OHG than OGG1-Ser326 in vivo in human cells; supporting the results of recent association studies that OGG1-Cys326 is a risk allele for several types of human cancers.

Topics: Adenoviridae; Blotting, Western; DNA Glycosylases; Genes, Suppressor; Guanine; Humans; Lung Neoplasms; Mutagenesis; Mutation; Plasmids; Polymorphism, Genetic; RNA, Transfer; Transfection; Tumor Cells, Cultured

To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with gamma-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10(7) guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10(7) Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10(7) dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10(7) Gua/Gy) was observed after cells were irradiated with gamma-rays. Twenty hours after gamma-irradiation with 60 Gy, 75% of the 8-OH-Gua produced in keratinocyte DNA was repaired. With our new analysis method, it is possible to detect the small changes in the 8-OH-Gua levels in cellular DNA induced by various environmental factors.

Topics: Cell Line; Cells; Chromatography, High Pressure Liquid; DNA; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Electrochemistry; Guanine; Humans; Keratinocytes; Lung Neoplasms; Oxidative Stress

Using Mth1 and Ogg1 knockout mice, we evaluated the roles of these enzymes to prevent tumorigenesis and the accumulation of 8-oxoguanine (8-oxoG) in DNA. We found that lung adenoma/carcinoma spontaneously developed in Ogg1 knockout mice approximately 1.5 years after birth in which 8-oxoG was found to accumulate in their genomes. The mean number of tumors/mouse was 0.71 for the Ogg1 knockout mice, which was five times higher than that observed in wild-type mice (0.14). Although the accumulation of 8-oxoG was also confirmed in the Ogg1, Mth1 double knockout mice, we found no tumor in the lungs of these mice. This observation suggests that Mth1 gene disruption resulted in a suppression of the tumorigenesis caused by an Ogg1 deficiency.

Topics: Adenocarcinoma; Animals; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; Female; Guanine; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Glycosyl Hydrolases; Pedigree; Phosphoric Monoester Hydrolases

In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20-60 Gy of gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA.

Topics: Biomarkers; Carcinoma; DNA Damage; DNA Repair; Epithelial Cells; Gamma Rays; Guanine; Guanosine Monophosphate; Humans; Lung Neoplasms; Oxidative Stress; Toxicity Tests; Tumor Cells, Cultured

Topics: Biomarkers, Tumor; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Environmental Exposure; Free Radicals; Genetic Predisposition to Disease; Guanine; Humans; Lung Neoplasms; N-Glycosyl Hydrolases; Neoplasms; Oxidative Stress; Phenotype; Polymorphism, Single Nucleotide; Research Design; Risk Factors; Smoking; Treatment Outcome

Although smoking is a major cause of lung cancer, only a proportion of smokers develop lung cancer, suggesting a genetic predisposition in some individuals. Because tobacco smoking is associated with the increased formation of DNA lesions, including those induced from oxidative damage, we investigated whether the activity of the DNA repair enzyme 8-oxoguanine DNA N-glycosylase (OGG), which repairs the oxidative DNA lesion 8-oxoguanine, is associated with lung cancer.. We conducted a molecular epidemiologic case-control study that included 68 case patients with non-small-cell lung cancer and 68 healthy control subjects, frequency matched for age and sex. Enzymatic OGG activity was determined in protein extracts prepared from peripheral blood mononuclear cells or lung tissue by assaying the cleavage product of a radiolabeled synthetic DNA oligonucleotide containing an 8-oxoguanine residue. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined by conditional logistic regression. All statistical tests were two-sided.. OGG activity was lower in peripheral blood mononuclear cells from case patients than in those from control subjects. After adjustment for age and smoking status, individuals in the lowest tertile of OGG activity had an increased risk of non-small-cell lung cancer compared with individuals in the highest tertile (OR = 4.8, 95% CI = 1.5 to 15.9). The adjusted OR associated with a unit decrease in OGG activity was statistically significantly increased (OR = 1.9, 95% CI = 1.3 to 2.8). There was no interaction between OGG activity and smoking status. The estimated relative risk of lung cancer for smokers with low OGG activity was 34- or 124-fold higher for smokers with a low OGG activity of 6.0 or 4.0 U/ micro g protein, respectively, than for nonsmokers with a normal OGG activity of 7.0 U/ micro g protein, illustrating the cumulative effect of low OGG activity and smoking.. Low OGG activity is associated with an increased risk of lung cancer. Although prospective studies are needed to validate the results, they suggest that smoking cessation in individuals with reduced OGG activity might be an effective strategy in lung cancer prevention.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Female; Guanine; Humans; Lung Neoplasms; Male; Middle Aged; N-Glycosyl Hydrolases; Odds Ratio; Oxidative Stress; Risk Assessment; Risk Factors; Smoking

Animal studies have demonstrated that ozone exposure can induce lung tumors. Recent epidemiological studies have also shown that increased ozone exposure is associated with a greater risk of lung cancer. This study used single-cell gel electrophoresis (the Comet assay) and flow cytometry to investigate DNA damage in A549 cells exposed to ozone levels below the current ambient standard. Cells were exposed to ozone at levels of 0, 60, 80, and 120 ppb, and then DNA single-strand breaks and 8-oxoguanine levels were measured. Additionally, the formamidopyrimidine glycosylase (Fpg) repair enzyme was added to the Comet assay to enhance detection of oxidative damage. Vitamins C and E were also added to determine their inhibitory effects on ozone-induced 8-oxoguanine. Measurements of tail length, tail intensity, and tail moment of the Comet assay were shown to correlate with each other. However, tail moment appeared to be more sensitive than the other two indicators in detecting DNA single-strand breaks. Tail moments of cells exposed to 80 and 120 ppb of ozone were significantly higher than those exposed to 0 ppb (P<0.05). These three indicators of DNA single-strand breaks with Fpg were shown to be increased and more sensitive than those without Fpg. After Fpg was introduced, the tail moments at ozone levels of 60, 80, and 120 ppb were significantly higher than those at 0 ppb (P<0.05). Furthermore, 8-oxoguanine levels, determined by fluorescence intensity, at 80 and 120 ppb of ozone exposure were significantly higher than the level at 0 ppb. Pretreatment with vitamins C and E reduced the 8-oxoguanine levels caused by ozone. We conclude that ozone levels below current ambient standards may induce DNA breaks and oxidative DNA damage. Moreover, the Fpg repair enzyme in the Comet assay can increase the sensitivity of oxidative damage detection in vitro.

Topics: Antioxidants; Ascorbic Acid; Comet Assay; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Dose-Response Relationship, Drug; Guanine; Humans; Lung Neoplasms; Oxidants, Photochemical; Oxidative Stress; Ozone; Tumor Cells, Cultured; Vitamin E

To elucidate the involvement of 8-hydroxyguanine (oh(8)G) repair genes in human lung carcinogenesis, 47 lung cancer cell lines and 55 primary lung cancers were examined for somatic mutations and genetic polymorphisms in all coding exons of the MYH and APEX genes, and exon 8 of the OGG1 gene by polymerase chain reaction-single strand conformation polymorphism analysis. In the MYH gene, one missense mutation was detected in a cell line, NCI-H157, whereas no mutations were detected in primary cancers. There were no mutations in the APEX and OGG1 genes in the cell lines or primary cancers. Ten single nucleotide polymorphisms (SNPs) were identified, and seven of them were accompanied by amino acid substitutions. Differences in the oh(8)G repair activities of MYH, APEX and OGG1 proteins due to somatic mutations and SNPs can be involved in human carcinogenesis.

Topics: Carbon-Oxygen Lyases; DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Guanine; Humans; Lung Neoplasms; Mutation; N-Glycosyl Hydrolases; Nuclear Proteins; Polymorphism, Single Nucleotide; Tumor Cells, Cultured

8-Hydroxyguanine (8-OHG) is an oxidatively damaged mutagenic base which causes G:C-->T:A transversions in DNA. OGG1 was cloned as a human gene encoding a DNA glycosylase that specifically excises 8-OHG from DNA in vitro. However, it was not clear whether OGG1 protein suppresses G:C-->T:A transversions caused by 8-OHG in human cells in vivo. In the present study we have examined the ability of OGG1 protein to suppress G:C-->T:A transversions caused by 8-OHG in human cells by bacterial suppressor tRNA (supF) forward mutation assay using a shuttle vector DNA, pMY189. Introduction of a single 8-OHG residue at position 159 of the supF gene in plasmid pMY189 resulted in a 130-fold increase in mutation frequency compared with untreated plasmid pMY189 after replication in the NCI-H1299 human lung cancer cell line. G:C-->T:A transversions at position 159 were detected in >90% of the supF mutants from the 8-OHG-containing plasmid. The mutation frequency of the 8-OHG-containing plasmid was significantly reduced by overexpression of OGG1 protein in NCI-H1299 cells and, in particular, the occurrence of G:C-->T:A transversion at position 159 in the supF gene was suppressed. Furthermore, frequencies and spectra of mutations of the untreated pMY189 plasmid did not differ significantly with overexpression of OGG1 protein. These results indicate that OGG1 protein has the ability to suppress G:C-->T:A transversions caused by 8-OHG in human cells in vivo.

Topics: Base Sequence; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Escherichia coli; Escherichia coli Proteins; Genes, Suppressor; Genetic Vectors; Guanine; Humans; Lung Neoplasms; Molecular Sequence Data; N-Glycosyl Hydrolases; Plasmids; Point Mutation; RNA, Transfer; Transfection; Tumor Cells, Cultured

The human homologue of the yeast OGG1 gene, hOGG1, has been cloned, and its genetic structure has been determined. Several polymorphisms in the hOGG1 gene were detected in the Japanese populations, and among them, the Ser-Cys polymorphism at codon 326 has been shown to have a functional difference in complementation of mutant Escherichia coli that is defective in the repair of 8-hydroxyguanine. Activity in the repair of 8-hydroxyguanine is greater in hOGG1-Ser326 protein than in hOGG1(326) protein. Because many environmental carcinogens produce 8-hydroxyguanine residue and mismatching to this modified base potentially causes oncogenic mutations, the capacity to repair these lesions can be involved in cancer susceptibility in human beings. We, therefore, examined allele distributions of the Ser326Cys polymorphism in a case-control study of male lung cancer in Okinawa. The analyses based on 241 cases and 197 hospital controls disclosed the following findings. (a) Those with the Cys/Cys genotype were at an increased risk of squamous cell carcinoma and nonadenocarcinoma compared to those with the Ser/Cys and those with the Ser/Ser genotypes combined. The odds ratios adjusted for age and smoking history were 3.01 (95% confidence interval, 1.33-6.83) and 2.18 (95% confidence interval, 1.05-4.54), respectively. (b) The odds ratios for other histological subtypes of lung cancer or those in total were not significant. Those for Cys/Cys or Ser/Cys genotype against Ser/Ser did not reach statistical significance in any cell type. (c) The distributions of this polymorphism varied for different populations (Chinese, Japanese, Micronesians, Melanesians, Hungarians, and Australian Caucasians), with much less prevalence of Cys allele in the latter three populations. Although our sample size was limited, these results indicate that the Ser326Cys variant may be related to squamous cell lung cancer susceptibility. The Cys/Cys genotype appears to be more susceptible to squamous cell carcinoma, although the risk is less than that previously reported to be associated with the CYP1A1 gene. Further studies are needed to assess the importance of the interpopulation variation to cancer susceptibility.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Carcinoma, Squamous Cell; Case-Control Studies; Cysteine; DNA Damage; Genetic Predisposition to Disease; Guanine; Humans; Japan; Lung Neoplasms; Male; Middle Aged; N-Glycosyl Hydrolases; Polymorphism, Genetic; Risk Assessment; Serine

Guanine residues in the genome are vulnerable to attack by free radicals and reactive oxygen species. A major lesion thus produced, 8-oxoguanine (OG), causes mutations by mis-pairing with adenine during replication. In bacteria and budding yeast, OG is removed from the genome through the action of base-excision DNA repair (BER) enzymes, which catalyze expulsion of the aberrant base and excision of its sugar moiety from the DNA backbone. Although OG is known to be produced in and cleansed from mammalian genomes, the enzymes responsible for OG repair in these cells have remained elusive.. Here, we report the cloning and biochemical characterization of mammalian BER enzymes that specifically target OG residues in DNA. These 8-oxoguanine DNA glycosylases, hOgg1 (human) and mOgg1 (murine), are homologous to each other and to yeast Ogg1. They also contain an active site motif - the Helix-hairpin-Helix, Gly/Pro-rich-Asp motif - characteristic of a superfamily of BER proteins with a similar core fold and active site geometry. Both hOgg1 and mOgg1 exhibit exquisite selectivity for the base opposite OG in DNA, operating with high efficiency only on OG base-paired to cytosine. Furthermore, hOgg1 and mOgg1 are unable to process a panel of alternative lesions, including 8-oxoadenine, yet bind with high affinity to synthetic abasic site analogs. The proteins operate through a classical glycosylase/lyase catalytic mechanism; mutation of a catalytically essential lysine residue results in loss of catalytic potency but retention of binding to OG-containing oligonucleotides. The hOGG1 gene is localized on the short arm of chromosome 3 (3p25/26) in a region commonly deleted in cancers.. These results conclusively establish the existence and identity of an 8-oxoguanine DNA glycosylase/lyase in human and murine cells, completing the triad of proteins that together protect mammals from the genotoxic effects of guanine oxidation. The observation that at least one allele of hOGG1 is commonly deleted in cancer cells suggests that such cells may possess a reduced capacity to counter the mutagenic effects of reactive oxygen species, a deficiency that could increase their overall genomic instability. This speculation is fueled by recent observations that cells constitutively active for the Ras/Raf pathway constitutively produce high levels of superoxide, a known generator of OG.

Topics: Amino Acid Sequence; Animals; Binding Sites; Chromosome Mapping; Cloning, Molecular; Deoxyadenosines; Deoxyguanosine; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Glycosylation; Guanine; Humans; In Situ Hybridization, Fluorescence; Lung Neoplasms; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases; Organophosphorus Compounds; Oxidation-Reduction; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Amino Acid; Substrate Specificity

Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ12) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Air Pollutants; Animals; Bronchoalveolar Lavage Fluid; Cell Division; Deoxyguanosine; Dust; Guanine; Image Processing, Computer-Assisted; Immunohistochemistry; Lung; Lung Neoplasms; Quartz; Rabbits; Rats; Rats, Wistar; Reactive Oxygen Species

The extent of DNA modification in cancerous rat live and lung tissues was investigated and compared to their respective normal tissues. Liver tumors were induced by 2-fluorenylacetamide (2-FAA) or N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA), and lung tumors were induced by sodium nitrite plus trimethylamine. In the DNA samples isolated from these tissues, two pyrimidine-derived and four purine-derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected-ion monitoring. These compounds were characterized as 5-hydroxyuracil (5-OHUra), thymine glycol (TG), 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5- formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-OHAde), and 8-hydroxyguanine (8-OHGua). Elevated amounts of modified DNA bases were found in most cancerous tissues when compared to the controls. Chemicals used for tumor induction were responsible for inducing DNA lesions that could be promutagenic in vivo and could lead to various types of mutations. When endogenous oxidative damage to DNA during aging was examined, a roughly 2-fold increase of thymine glycol, 8-OHAde and 8-OHGua was found in aged (12 months) rat liver tissues compared to young tissues (1 month). The same results were also found in lung tissues, except that the amount of thymine glycol exhibited more than a 10-fold increase in aged tissues when compared to young tissues. The association of the modified bases with the processes of aging and carcinogenesis deserves further investigation.

Topics: 2-Acetylaminofluorene; Adenine; Aging; Animals; Antineoplastic Agents; Disease Models, Animal; DNA Damage; DNA, Neoplasm; Gas Chromatography-Mass Spectrometry; Guanine; Hydrolysis; Liver; Liver Neoplasms, Experimental; Lung; Lung Neoplasms; Male; Methylamines; Nitrates; Oxidation-Reduction; Pyrimidines; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Thymine; Uracil