A limited association of OGG1 Ser326Cys polymorphism for adenocarcinoma of the lung

The 8-oxoguanine DNA glycosylase (OGG1) repairs DNA by removing 8-hydroxyguanine, a highly mutagenic oxidative DNA adduct. Recently, the gene for OGG1 was cloned and several polymorphisms have been reported. Because environmental carcinogens produce 8-hydroxyguanine residues that potentially cause oncogenic mutations by mismatching to this modified base, the capacity to repair these lesions can be involved in cancer susceptibility. This study investigated the association between OGG1 Ser326Cys polymorphism and risk of the lung adenocarcinoma for Japanese by a prevalent case-control study in Japan. The subjects comprised 138 cases and 241 non-cancer outpatients as controls. OGG1 gene polymorphism was genotyped by a PCR-CTPP (polymerase chain reaction with confronting two-pair primers) method. The distribution of OGG1 Ser326Cys genotype among controls (Ser/Ser, 28.3%; Ser/Cys, 49.2%; and Cys/Cys, 22.5%) was not different from that among cases (Ser/Ser, 29.0%; Ser/Cys, 51.4%; and Cys/Cys, 24.0%). The sex-age adjusted odds ratio (OR) was 1.06 with 95% confidence interval (CI) 0.64-1.76 for Ser/Cys genotype and 0.81 with 0.44-1.52 for Cys/Cys genotype. The ORs according to the interval between diagnosis and study enrollment were also examined because the polymorphism was a potential prognostic factor of lung cancer. The ORs of Ser/Cys and Cys/Cys genotypes in the cases less than 3 years after diagnosis were higher than overall ORs; 1.86 (95%CI, 0.91-3.77), and 1.46 (0.64-3.35), respectively. The OR for smoking was not statistically different among genotype, though the sample size was too small to detect even a moderate interaction. This study supported the first study by Sugimura et al (Cancer Epidemiol Biomarkers Prev, 1999; 8: 669-674), that the association of OGG1 Ser326Cys polymorphism was limited for the risk of lung adenocarcinoma.     

Generation, biological consequences and repair mechanisms of cytosine deamination in DNA

Base moieties in DNA are spontaneously threatened by naturally occurring chemical reactions such as deamination, hydrolysis and oxidation. These DNA modifications have been considered to be major causes of cell death, mutations and cancer induction in organisms. Organisms have developed the DNA base excision repair pathway as a defense mechanism to protect them from these threats. DNA glycosylases, the key enzyme in the base excision repair pathway, are highly conserved in evolution. Uracil constantly occurs in DNA. Uracil in DNA arises by spontaneous deamination of cytosine to generate pro-mutagenic U:G mispairs. Uracil in DNA is also produced by the incorporation of dUMP during DNA replication. Uracil-DNA glycosylase (UNG) acts as a major repair enzyme that protects DNA from the deleterious consequences of uracil. The first UNG activity was discovered in E. coli in 1974. This was also the first discovery of base excision repair. The sequence encoded by the ung gene demonstrates that the E. coli UNG is highly conserved in viruses, bacteria, archaea, yeast, mice and humans. In this review, we will focus on central and recent findings on the generation, biological consequences and repair mechanisms of uracil in DNA and on the biological significance of uracil-DNA glycosylase.     

Alcohol consumption and oxidative DNA damage

To examine the effects of alcohol consumption on cancer risk, we measured oxidative DNA damage and its repair activity in the livers and esophagi of rats fed with ethanol. Using our previously designed protocol for feeding rats with a high concentration of ethanol, we examined the effects of ethanol consumption on 8-oxo-Gua generation and repair activity in the livers and esophagi of rats. We found that a high concentration of ethanol accompanied with a vitamin-depleted diet increased 8-oxo-Gua and its repair activity. 8-Oxo-Gua is known to induce point mutations, leading to carcinogenesis. Therefore, these results suggested that a high concentration of ethanol and an irregular diet increased liver and esophageal cancer risk. On the other hand, we showed that a low concentration of ethanol decreased 8-oxo-Gua and its repair activity in the livers of mice treated with a carcinogen. Taken together, the effects of ethanol consumption on cancer risk depend on the ethanol concentration and the diet pattern.     

Analysis of 8-hydroxyguanine (8-OH-Gua) released from DNA by the formamidopyrimidine DNA glycosylase (Fpg) protein: a reliable method to estimate cellular oxidative stress

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.     

The detection of pollutant impact in marine environments: Condition index, oxidative DNA damage, and their associations with metal bioaccumulation in the Sydney rock oyster Saccostrea commercialis

Specimens of the Sydney rock oyster Saccostrea commercialis were deployed for a 3-month period at control and sewage disturbed marine locations in the Hunter Region, New South Wales, Australia. The DNA damage product, 8-hydroxyguanine, was measured by GC/MS-SIM from chromatin extracts of the gill tissues of oysters to assess oxidative damage. The levels ranged from 11.5 to 18.8 modified bases per 10⁷ guanine bases. Although the condition indices were significantly different between the Redhead control site (178.3±3.6) and the Burwood sewage disturbed location (140.4±4.4), no significant differences in 8-hydroxyguanine concentrations were detected between the sites, and the concentration of 8-hydroxyguanine was not correlated to condition index. However, levels of the DNA base modification were correlated with the concentrations of bioaccumulated lead (r=0.84, P=0.036). This association provides in vivo evidence that the bioaccumulation of lead results in oxidative damage to DNA. An additional control and sewage disturbed site were included to investigate the relationship between heavy metal bioaccumulation and the condition index of deployed oysters. After the 3-month deployment period, the condition index was negatively correlated to concentrations of bioaccumulated mercury (r=–0.80, P<0.001), cobalt (r=–0.65, P<0.01), and nickel (r=–0.69, P<0.01), suggesting a strong negative influence of these metals at relatively low concentrations on the physiological condition of the oysters.     

The Shizosaccharomyces pombe homolog (SpMYH) of the Escherichia coli MutY is required for removal of guanine from 8-oxoguanine/guanine mispairs to prevent G:C to C:G transversions

The frequency of G:C-->C:G transversions significantly increases upon exposure of cells to ionizing radiation or reactive oxygen species. Transversions can be prevented by base excision repair, which removes the causative modified bases from DNA. Our previous studies revealed that MutY is responsible for removing guanine from 7,8-dihydro-8-oxoguanine/guanine mispairs (8-oxoG/G) and prevents the generation of G:C-->C:G transversions in E. coli. SpMYH, a homolog of E. coli MutY, had been identified and characterized in the fission yeast S. pombe. Purified SpMYH has adenine DNA glycosylase activity on A/8-oxoG and A/G mismatch-containing oligonucleotides. In this study, we examined whether SpMYH has a similar activity allowing it to remove G from 8-oxoG/G in DNA. The purified SpMYH tightly bound to duplex oligonucleotides containing 8-oxoG/G and removed the unmodified G from 8-oxoG/G as efficiently as A from 8-oxoG/A. The activity was absent in the cell extract prepared from an SpMYH-knockout strain of S. pombe. The expression of SpMYH markedly reduced the frequency of spontaneous G:C-->C:G transversions in the E. coli mutY mutant. These results demonstrate that SpMYH is involved in the repair of 8-oxoG/G, by which it prevents mutations induced by oxidative stress in S. pombe.     

Recombinant Schizosaccharomyces pombe Nth1 protein exhibits DNA glycosylase activities for 8-oxo-7,8-dihydroguanine and thymine residues oxidized in the methyl group

Bacteria and eukaryotes possess redundant enzymes that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases remove damaged bases to initiate the base excision repair. The exocyclic methyl group of thymine does not escape oxidative damage to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). 5-foU is a potentially mutagenic lesion. A homolog of E. coli endonuclease III (SpNth1) had been identified and characterized in Schizosaccharomyces pombe. In this study, we found that SpNth1 recognizes and removes 5-foU and 5-hmU from DNA with similar efficiency. The specific activities for the removal of 5-foU and 5-hmU were comparable with that for thymine glycol. The expression of SpNth1 reduced the hydrogen peroxide toxicity and the frequency of spontaneous mutations in E. coli nth nei mutant. It was also revealed that SpNth1 had DNA glycosylase activity for removing 8-oxo-7,8-dihydroguanine (8-oxoG) from 8-oxoG/G and 8-oxoG/A mispairs. These results indicated that SpNth1 has a broad substrate specificity and is involved in the base excision repair of 8-oxoG and thymine residues oxidized in the methyl group in S. pombe.     

High dietary intake of sodium selenite induces oxidative DNA damage in rat liver

One mechanism for the cancer-chemopreventive effects of high selenium (Se) intake is hypothesized to be antioxidant protection of DNA. In this work we examine DNA oxidation in whole animals as a function of dietary Se intake and carcinogen administration. Weanling male Sprague-Dawley rats were fed a basal, Torula yeast-based, Se-deficient diet supplemented with 0, 0.15, or 2.0 ppm Se as sodium selenite for 10 wk. They were then injected with 0, 0.1, or 10 mg /kg body weight of the pro-oxidant carcinogen N-nitrosodiethylamine. High levels of carcinogen and high levels of selenite intake each increased concentration of 8-hydroxy-2'-deoxyguanosine in liver DNA. Se-dependent glutathione peroxidase I gene expression and enzyme activity were dramatically reduced by dietary Se deficiency but were unaffected by carcinogen administration. There were no significant main or interactive effects of Se or carcinogen on activity or gene expression of the DNA repair enzyme 8-oxoguanine glycosylase I. These results do not support the hypothesis that high Se intake may be cancer-preventive by inhibiting oxidative DNA damage. Rather than inhibiting oxidative DNA damage, these findings suggest that high dietary intake of inorganic Se may promote in vivo DNA oxidation.     

The effects of 1-nitropyrene on oxidative DNA damage and expression of DNA repair enzymes

Nitropyrenes (NPs) present in diesel and gasoline emissions are mutagenic and carcinogenic in experimental animals. Nitro-reduction of 1-NP causes oxidative stress. It is unclear whether 8-hydroxydeoxyguanosine (8-OH-dG) is produced from 1-NP and whether it contributes to the carcinogenic activity of 1-NP. In this study, we measured the level of reactive oxygen species (ROS) in cultured human lung epithelial cells after exposure to 1-NP and the intracellular level of 8-OH-dG and expression level of the 8-OH-dG repair enzymes. As results, 1-NP induced the generation of 8-OH-dG via ROS, but 8-OH-dG repair enzymes prevented an increase of 8-OH-dG formation in cellular DNA of the A549 cell line below 250 microM of 1-NP. These data suggest that 1-NP can induce oxidative DNA damage by generation of ROS, which may play a role in the carcinogenesis induced by 1-NP. These data also suggest that individuals with impaired DNA repair enzymes might be more susceptible to lung cancer induced by 1-NP.     

DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites

Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.     

Production and repair of DNA damage in mammalian cells

Cellular genomes are continually subjected to endogenous and environmentally induced structural alterations. Our environment contains a multitude of substances that are carcinogenic and which, in many cases, are thought to act via direct damage to DNA. In order to better understand the consequences of DNA damage in cell killing and carcinogenesis, it will be important to: (a) develop new and sensitive techniques for the identification of specific types of DNA lesions; (b) examine what influence the function or activity of a DNA sequence has on the distribution of DNA damage within that DNA sequence and on its repair efficiency; (c) examine what influence the cell cycle has on the efficiency of DNA repair.     

Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge

Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.     

Adenine Glycosylase MutY of Corynebacterium pseudotuberculosis presents the antimutator phenotype and evidences of glycosylase/AP lyase activity in vitro

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe-4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.     

Repair system of 7, 8-dihydro-8-oxoguanine as a defense line against carcinogenesis

Reactive oxygen species (ROS) are essentially harmful for living organisms, including human beings. It is well known that ROS-induced damage of cellular components may lead to human diseases, such as inflammatory diseases, degenerative diseases, or cancer. In particular, oxidative DNA damage is premutagenic, and thus, the generation of DNA damage and the failure of its removal are critical events for tumorigenesis or carcinogenesis. To prevent this disadvantage, living organisms have defense mechanisms against ROS-induced gene instability. Studies of 8-oxo-Gua and its main repair enzyme, 8-oxoguanine DNA glycosylase 1 (OGG1), are informative and useful, because 8-oxo-Gua is commonly observed in DNA, and OGG1 enzymes exist in a wide variety of living organisms. The importance of OGG1 was confirmed by polymorphism analyses and studies using knockout mice. Moreover, analyses of the influences of environmental factors on DNA damage and repair systems have confirmed the effects of heavy metals on 8-oxo-Gua formation and OGG1 expression. These studies revealed that the 8-oxo-Gua repair system is crucial for the prevention of mutation-related diseases, such as cancer. In this review, the advances in this field during the last two decades are described.     

Ethanol modulates rat hepatic DNA repair functions

To further explore how ethanol may act at the DNA level, studies have been made of DNA repair mechanisms in male Wistar rats given ethanol either as an acute intragastric dose (5 g/kg) or continuously in a liquid diet (5% w/v) to provide 36% of the caloric intake. These treatments generate significant levels of free radicals with evidence of damage to DNA. The acute ethanol dose significantly inhibited O(6)-alkylguanine-DNA alkyltransferase (ATase) activity by 21-32% throughout the 24-h post-treatment period and this was confirmed by immunohistochemical detection of the ATase protein in hepatic nuclei. Twelve hours after the ethanol treatment, the activities of the DNA glycosylases, alkylpurine-DNA-N-glycosylase (APNG) and 8-oxoguanine-DNA glycosylase (OXOG glycosylase) were each increased by approximately 44%. In contrast, when given chronically via the liquid diet, ethanol initially had no effect on ATase activity, but after 4 weeks ATase activity was increased by 40%. Following ethanol withdrawal, ATase activity remained elevated for at least 12 h, but, by 24 h, the activity had fallen to the uninduced control level. DNA glycosylase activities were again affected differently. After 1 week of dietary ethanol exposure, there was no effect on APNG activity but it was inhibited by 19% at 4 weeks. OXOG glycosylase activity, on the other hand, was increased by 53% after 1 week, but decreased by 40% after 4 weeks. Although some of these changes in DNA repair capacity were relatively small, over time, their potential impact on the repair of endogenous or exogenous alkylation and/or oxidation damage in DNA would be substantial. These studies indicate possible mechanisms for the co-carcinogenic effects of ethanol.     

Oxidatively Damaged DNA in Rats Exposed by Oral Gavage to C60 Fullerenes and Single-Walled Carbon Nanotubes

Background

C₆₀ fullerenes and single-walled carbon nanotubes (SWCNT) are projected to be used in medicine and consumer products with potential human exposure. The hazardous effects of these particles are expected to involve oxidative stress with generation of oxidatively damaged DNA that might be the initiating event in the development of cancer.

Objective

In this study we investigated the effect of a single oral administration of C₆₀ fullerenes and SWCNT.

Methods

We measured the level of oxidative damage to DNA as the premutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in the colon mucosa, liver, and lung of rats after intragastric administration of pristine C₆₀ fullerenes or SWCNT (0.064 or 0.64 mg/kg body weight) suspended in saline solution or corn oil. We investigated the regulation of DNA repair systems toward 8-oxodG in liver and lung tissue.

Results

Both doses of SWCNT increased the levels of 8-oxodG in liver and lung. Administration of C₆₀ fullerenes increased the hepatic level of 8-oxodG, whereas only the high dose generated 8-oxodG in the lung. We detected no effects on 8-oxodG in colon mucosa. Suspension of particles in saline solution or corn oil yielded a similar extent of genotoxicity, whereas corn oil per se generated more genotoxicity than the particles. Although there was increased mRNA expression of 8-oxoguanine DNA glycosylase in the liver of C₆₀ fullerene-treated rats, we found no significant increase in repair activity.

Conclusions

Oral exposure to low doses of C₆₀ fullerenes and SWCNT is associated with elevated levels of 8-oxodG in the liver and lung, which is likely to be caused by a direct genotoxic ability rather than an inhibition of the DNA repair system.     

Purification and characterization of a novel DNA repair enzyme from the extremely radioresistant bacterium Rubrobacter radiotolerans

Rubrobacter radiotolerans is an extremely radioresistant bacterium. It exhibits higher resistance than the well-known radioresistant bacterium Deinococcus radiodurans, but the molecular mechanisms responsible for the radio-resistance of R. radiotolerans remain unknown. In the present study, we have demonstrated the presence of a novel DNA repair enzyme in R. radiotolerans cells that recognizes radiation-induced DNA damages such as thymine glycol, urea residues, and abasic sites. The enzyme was purified from the crude cell extract by a series of chromatography to an apparent physical homogeneity. The purified enzyme showed a single band with a molecular mass of approximately 40 kDa in SDS-polyacrylamide gel electrophoresis, and was designated as R-endonuclease. R-Endonuclease exhibited repair activity for thymine glycol, urea residues, and abasic sites present in plasmid DNA, but did not act on intact DNA, UV-irradiated DNA and DNA containing reduced abasic sites. The substrate specificity together with the salt and pH optima suggests that R-endonuclease is a functional homolog of endonuclease III of Escherichia coli.     

Polar, functionalized guanine-O6 derivatives resistant to repair by O6-alkylguanine-DNA alkyltransferase: implications for the design of DNA-modifying drugs

The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.