Mitochondrial endogenous oxidative damage has been overestimated.

The oxidatively induced DNA lesion 8-oxo-dG in mitochondrial DNA (mtDNA) is commonly used as a marker for oxidative damage to mitochondria, which in turn is thought to be a fundamental cause of aging. For years, mitochondrial levels of 8-oxo-dG were believed to be approximately 10-fold higher in mtDNA than in nuclear DNA even in normal, young animals. However, studies in our own and other laboratories have shown that this lesion is efficiently repaired. Also, mutational consequences specific to 8-oxo-dG (G to T transversions) are rarely reported. In the present study, we showed that the levels of damage measured using high-pressure liquid chromatography/electrochemical detection and an enzymatic/Southern blot assay were comparable. The latter assay does not require isolation of mitochondria, and so this assay was then used to determine the level of in vivo damage present in rat liver mtDNA both with and without organelle isolation. Levels of 8-oxo-dG are approximately threefold higher when measured in mtDNA purified from isolated mitochondria than when measured without prior mitochondrial isolation. Furthermore, most genomes were free of endogenous enzyme-sensitive sites (i.e., they did not contain 8-oxo-dG), and only after mitochondrial isolation were levels higher in mtDNA than in a nuclear sequence. Anson, R. M., Hudson, E., Bohr, V. A. Mitochondrial endogenous oxidative damage has been overestimated.     

Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS

A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding 15N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.     

Regulation of a novel cell differentiation-associated gene,JWA during oxidative damage in K562 and MCF-7 cells

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothesized as the major source of DNA damage that can lead to a variety of diseases including cancer. It is known that 8-hydroxy-deoxyguanosine (8-oxo-dG) is a useful biomarker of oxidative DNA damage. Our recent data showed that JWA, initially being cloned as a novel cell differentiation-associated gene, was also actively responsive to environmental stressors, such as heat-shock, oxidative stress and so on. In the present study, we have applied a modified comet assay and bacterial repair endonucleases system (endonuclease III and formamidopyrimidine glycosylase) to investigate if JWA is involved in hydrogen peroxide (H₂O₂)-induced DNA damage and repair in K562 and MCF-7 cells, and to demonstrate if the damage is associated with 8-oxo-dG. The results from the comet assay have shown that the average tail length and the percentage of the cells with DNA tails are greatly induced by H₂O₂ treatment and further significantly enhanced by the post-treatment of repair endonucleases. The H₂O₂-induced 8-oxo-dG formation in K562 and MCF-7 cells is dose-dependent. In addition, the data have clearly demonstrated that JWA gene expression is actively induced by H₂O₂ treatment in K562 and MCF-7 cells. The results suggest that JWA can be regulated by oxidative stress and is actively involved in the signal pathways of oxidative stress in the cells.     

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.     

Attenuation of neointima formation through the inhibition of DNA repair enzyme PARP-1 in balloon-injured rat carotid artery

Increased oxidative stress is a major characteristic of restenosis after angioplasty. The oxidative stress is mainly created by oxidants such as reactive oxygen species (ROS), which are assumed to play an important role in neointima formation after angioplasty. DNA is a sensitive target for oxidants; however, oxidative DNA damage remains a poorly examined field in the pathogenesis of restenosis. In the present study, we demonstrated that the expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was quickly increased in rat carotid arteries after balloon injury. It reached its peak at 14 days after injury and still kept high expression at 28 days after injury. The immunostaining of 8-oxo-dG was present predominantly in the neointima. In response to oxidative DNA damage, the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was significantly increased after balloon injury. The time course change and location of PARP-1 is similar to that of 8-oxo-dG. Daily injections of the PARP-1 inhibitor PJ34 (5 mg.kg(-1).day(-1) ip) attenuated neointima formation by approximately 40% at 7, 14, and 28 days after balloon injury. Treatment with PJ34 inhibited leukocyte infiltration and improved both anatomic (reendothelialization) and functional (endothelial function) recovery of endothelial cells after balloon injury. In conclusion, levels of oxidative DNA damage and the DNA repair enzyme PARP-1 are increased in vessels after balloon injury. Inhibition of PARP-1 attenuates neointima formation through inhibition of leukocyte infiltration and improvement of endothelial cell recovery after balloon injury. Targeting of the DNA repair enzyme might be a therapeutic strategy for restenosis.     

Epigallocatechin gallate markedly enhances formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the reaction of 2'-deoxyguanosine with hypochlorous acid

A tea polyphenol, (-)-epigallocatechin gallate (EGCG), which can scavenge a variety of reactive oxygen species, enhances the yield of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) up to 20-fold in the reaction of 2'-deoxyguanosine with hypochlorous acid (HOCl), compared with the reaction without EGCG. Certain concentrations of EGCG inhibited HOCl-mediated oxidation of 2'-deoxyguanosine to 8-oxo-dG to a limited extent, but efficiently inhibited further oxidation of 8-oxo-dG to spiroiminodihydantoin nucleoside, resulting in the accumulation of 8-oxo-dG in the reaction mixture. Conversely, EGCG inhibited dose-dependently an increase in 8-oxo-dG levels in calf thymus DNA incubated with HOCl. However, addition of HOCl to the DNA preoxidized with an oxidant-generating system (CuCl2, ascorbate, H2O2), led to the extensive loss of 8-oxo-dG due to its further oxidation. EGCG effectively inhibited this HOCl-mediated loss of 8-oxo-dG in the oxidized DNA, resulting in an apparent increase in 8-oxo-dG levels in the oxidized DNA, compared with the levels found without EGCG. The conversion of 8-oxo-dG into other oxidized lesions will inevitably affect recognition by DNA repair enzymes as well as the rates of mutations and DNA synthesis. Thus, our results suggest that as a biomarker of oxidative DNA damage, not only 8-oxo-dG but also the products of its further oxidation should be analyzed.     

Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine.

The initial aim of this study was to investigate how charge and other chemical properties of some radical scavengers influence the radiation-induced formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in two model systems. The target molecule, deoxyguanosine (dG), was either organized in the DNA-helix form or present as a free nucleoside in an aerated aqueous phosphate buffer. Samples were irradiated with 137Cs gamma rays, alone or in the presence of different thiols, alcohols or ascorbate with net charges from -1 to +1. The formation of 8-oxo-dG was assayed with reverse-phase HPLC coupled to an electrochemical detector. In the absence of radical scavengers, the radiation-induced formation of 8-oxo-dG in DNA was extensive, and the ratio for formation of 8-oxo-dG was 20-fold higher for DNA compared to dG. The yields of 8-oxo-dG in DNA and dG were 7.7 x 10(-3) micromol J(-1) and 3.8 x 10(-4) micromol J(-1), respectively. Yield-dose plots showed that the efficiency of the positively charged thiol cysteamine to counteract the radiation-induced formation of 8-oxo-dG in DNA was significantly (P < 0.001) greater compared to the uncharged or negatively charged thiols. Uncharged thiols were significantly (0.001 < P < 0.05) more effective in protecting DNA compared to negatively charged thiols. In contrast to the protection against oxidative damage provided by thiols and ascorbate when they were present during irradiation of DNA, the formation of 8-oxo-dG was significantly increased when these compounds were present during irradiation of dG in solution. Compared to the irradiated control, the increase was 11- to 116-fold for thiols and ascorbate, respectively. The enhanced oxidative damage of dG observed in the presence of ascorbate or thiols suggests that secondarily formed radicals from thiols or ascorbate may react with dG, or that transformation of different primary sites of damage on dG to 8-oxo-dG is enhanced.     

G-to-T transversions and small tandem base deletions are the hallmark of mutations induced by ultraviolet a radiation in mammalian cells

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm(2) produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage.     

8-Oxoguanine induces intramolecular DNA damage but free 8-oxoguanine protects intermolecular DNA from oxidative stress

7,8-Dihydro-8-oxoguanine (8-oxoguanine; 8-oxo-G), one of the major oxidative DNA adducts, is highly susceptible to further oxidation by radicals. We confirmed the higher reactivity of 8-oxo-G toward reactive oxygen (singlet oxygen and hydroxyl radical) or nitrogen (peroxynitrite) species as compared to unmodified base. In this study, we raised the question about the effect of this high reactivity toward radicals on intramolecular and intermolecular DNA damage. We found that the amount of intact nucleoside in oligodeoxynucleotide containing 8-oxo-G decreased more by various radicals at higher levels of 8-oxo-G incorporation, and that the oligodeoxynucleotide damage and plasmid cleavage by hydroxyl radical were inhibited in the presence of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG). We conclude that 8-oxo-G within DNA induces intramolecular DNA base damage, but that free 8-oxo-G protects intermolecular DNA from oxidative stress. These results suggest that 8-oxo-G within DNA must be rapidly released to protect DNA from overall oxidative damage.     

Mitochondrial GPx1 decreases induced but not basal oxidative damage to mtDNA in T47D cells

The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 microM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.     

2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA

8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.     

8-Oxo-7,8-dihydro-2'-deoxyguanosine is not salvaged for DNA synthesis in human leukemic U937 cells

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), the most common oxidatively modified nucleoside, is released from oxidized DNA and oxidized nucleotide pool. However, little information is available regarding the metabolic pathway of free 8-oxo-dG. In this study, we generated radiolabeled 8-oxo-dG to track its metabolic fate. We report that 8-oxo-dG is neither phosphorylated to 8-oxo-dGMP nor degraded to the free base, 8-oxo-7,8-dihydroguanine (8-oxo-Gua), indicating that 8-oxo-dG is not a substrate for nucleotide synthesis. This result was confirmed by the finding that no radioactivity was detected in the DNA of U937 cells after incubating the cells with radiolabeled 8-oxo-dG. These observations indicate that 8-oxo-dG produced by oxidative stress is not reutilized for DNA synthesis.     

Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension

The potential use of oxidative stress products as disease markers and progression is an important aspect of biomedical research. In the present study, the quantification of urine 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) concentration has been used to express the oxidation status of hypertensive subjects.

8-oxo-dG has been simultaneously isolated and assayed in nuclear (nDNA) and mitochondrial DNA (mtDNA). In addition, oxidative stress of mononuclear cells has been estimated by means of GSH and GSSG levels and GSSG/GSH ratio in hypertensive subjects before and after antihypertensive treatment. It is shown that oxidative stress decreases significantly in hypertensive patients after treatment the effect being accompanied by reduction of their blood pressure.

A significant correlation is observed comparing the yield of urine 8-oxo-dG and that isolated from mitochondria DNA. Moreover, urinary excretion of 8-oxo-dG also correlates with the GSSG/GSH ratio of cells. Conclusion: urine 8-oxo-dG assay is a good marker for monitoring oxidative stress changes in hypertensives.     

Senescence-associated decline in the intranuclear accumulation of hOGG1-alpha and impaired 8-oxo-dG repair activity in senescing normal human oral keratinocytes in vivo

We determined the mitochondrial membrane status, presence of reactive oxygen species (ROS), and oxidative DNA adduct formation in normal human oral keratinocytes (NHOK) during senescence. The senescent cells showed accumulation of intracellular ROS and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), a major oxidative DNA adduct. Exposure of cells to H2O2 induced 8-oxo-dG accumulation in cellular DNA, which was rapidly removed in replicating NHOK. However, the 8-oxo-dG removal activity was almost completely abolished in the senescing culture. Both replicating and senescing NHOK expressed readily detectable 8-oxo-dG DNA glycosylase (hOGG1), the enzyme responsible for glycosidic cleavage of 8-oxo-dG. After exposure to H2O2, however, the intranuclear level of the hOGG1-alpha isoform was decreased in senescing but not in replicating NHOK. These results indicated that senescing NHOK accumulated oxidative DNA lesions in part due to increased level of endogenous ROS and impaired intranuclear translocation of hOGG1 enzyme upon exposure to oxidative stress.     

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.     

Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in vivo and in vitro

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5-1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension

The potential use of oxidative stress products as disease markers and progression is an important aspect of biomedical research. In the present study, the quantification of urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) concentration has been used to express the oxidation status of hypertensive subjects.

8-oxo-dG has been simultaneously isolated and assayed in nuclear (nDNA) and mitochondrial DNA (mtDNA). In addition, oxidative stress of mononuclear cells has been estimated by means of GSH and GSSG levels and GSSG/GSH ratio in hypertensive subjects before and after antihypertensive treatment. It is shown that oxidative stress decreases significantly in hypertensive patients after treatment the effect being accompanied by reduction of their blood pressure.

A significant correlation is observed comparing the yield of urine 8-oxo-dG and that isolated from mitochondria DNA. Moreover, urinary excretion of 8-oxo-dG also correlates with the GSSG/GSH ratio of cells. Conclusion: urine 8-oxo-dG assay is a good marker for monitoring oxidative stress changes in hypertensives.     

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (k_pol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner''s syndrome cells.     

"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.