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DNA oxidation is a serious threat to genome integrity and is involved in mutations and cancer initiation. The G base is most frequently damaged, and 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the predominant damaged bases. In human cells, GO causes a G:C→T:A transversion mutation at the modified site, and also induces untargeted substitution mutations at the G bases of 5ʹ-GpA-3ʹ dinucleotides (action-at-a-distance mutations). The 5ʹ-GpA-3ʹ sequences are complementary to the 5ʹ-TpC-3ʹ sequences, the preferred substrates for apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) cytosine deaminases, and thus their contribution to mutagenesis has been considered. In this study, APOBEC3B, the most abundant APOBEC3 protein in human U2OS cells, was knocked down in human U2OS cells, and a GO-shuttle plasmid was then transfected into the cells. The action-at-a-distance mutations were reduced to ~25% by the knockdown, indicating that GO-induced action-at-a-distance mutations are highly dependent on APOBEC3B in this cell line. [ABSTRACT FROM AUTHOR]

In this proof-of-concept study, gold nanoparticles (AuNPs) were immobilized on glassy carbon electrode (GCE) surfaces using a surface-anchored diazonium salt of 4-aminothiophenol (GCE-Ph-S-AuNPs). X-ray photoelectron spectroscopy (XPS) studies confirmed the attachment of the AuNPs via 4-thiophenol onto the surface of the modified electrode. Differential pulse voltammetry (DPV) was performed for the simultaneous determination of guanine (G) and 8-hydroxyguanine (8-OH-G). The calibration curves were linear up to 140 µM and 60 µM with a limit of detection of 0.02 µM and 0.021 µM for G and 8-OH-G, respectively. Moreover, chronoamperometric studies were carried out for the determination of diffusion coefficients of 8-OH-G and G. The GCE-Ph-S-AuNPs were also applied in genomic DNA-spiked samples for the determination of G and 8-OH-G with recovery rates between 98.5% and 103.3%. The novel electrochemical surface provided a potential platform for the sensitive detection of 8-OH-G related to oxidative stress-induced DNA damage in clinical studies. [ABSTRACT FROM AUTHOR]

Purpose Cellular RNA is less compact than DNA, more easily accessible to ROS and therefore could be more susceptible to oxidative damage. This study was conceived in order to analyze the RNA oxidative damage in the urine of patients undergoing operation for colorectal cancer (CRC), to compare with healthy controls, and correlate with the stage. Materials and methods The study population was constituted by a group of 147 patients and a group of 128 healthy controls. Urine and blood samples were collected before the colonoscopy in all participants and 24 hours post-operatively for those who underwent surgery. Urine 8-hydroxyguanine (8-OHG) was determined as marker of RNA oxidation, and serum uric acid (UA) as antioxidant marker. Results Preoperatively, 8-OHG (ng/ml) values of CRC patients were found to be significantly higher than those of controls (p = 0.001). More specifically, stages II/III had significantly higher 8-OHG values (p

Numerous reports find that Cd induces formation of reactive oxygen species (ROS) in plants. However, a general ROS pool is usually studied, without distinction of their production site. In the present study, we applied a mitochondria-specific antioxidant, MitoTEMPO, to elucidate the role of mitochondria-derived ROS in the response of soybean seedlings to short-term (48 h) Cd stress. The obtained results showed that Cd caused a reduction in root length and fresh weight and increase in the level of superoxide anion, hydrogen peroxide, markers of lipid peroxidation (thiobarbituric reactive substances, TBARS) and markers of RNA oxidation (8-hydroxyguanosine, 8-OHG) in seedling roots. Application of MitoTEMPO affected Cd uptake in a dose-dependent manner and diminished the Cd-dependent induction of superoxide anion and lipid peroxidation. [ABSTRACT FROM AUTHOR]

In this proof-of-concept study, gold nanoparticles (AuNPs) were immobilized on glassy carbon electrode (GCE) surfaces using a surface-anchored diazonium salt of 4-aminothiophenol (GCE-Ph-S-AuNPs). X-ray photoelectron spectroscopy (XPS) studies confirmed the attachment of the AuNPs via 4-thiophenol onto the surface of the modified electrode. Differential pulse voltammetry (DPV) was performed for the simultaneous determination of guanine (G) and 8-hydroxyguanine (8-OH-G). The calibration curves were linear up to 140 µM and 60 µM with a limit of detection of 0.02 µM and 0.021 µM for G and 8-OH-G, respectively. Moreover, chronoamperometric studies were carried out for the determination of diffusion coefficients of 8-OH-G and G. The GCE-Ph-S-AuNPs were also applied in genomic DNA-spiked samples for the determination of G and 8-OH-G with recovery rates between 98.5% and 103.3%. The novel electrochemical surface provided a potential platform for the sensitive detection of 8-OH-G related to oxidative stress-induced DNA damage in clinical studies.

Cellular RNA is less compact than DNA, more easily accessible to ROS and therefore could be more susceptible to oxidative damage. This study was conceived in order to analyze the RNA oxidative damage in the urine of patients undergoing operation for colorectal cancer (CRC), to compare with healthy controls, and correlate with the stage. The study population was constituted by a group of 147 patients and a group of 128 healthy controls. Urine and blood samples were collected before the colonoscopy in all participants and 24 hours post-operatively for those who underwent surgery. Urine 8-hydroxyguanine (8-OHG) was determined as marker of RNA oxidation, and serum uric acid (UA) as antioxidant marker. Preoperatively, 8-OHG (ng/ml) values of CRC patients were found to be significantly higher than those of controls (p = 0.001). More specifically, stages II/III had significantly higher 8-OHG values (p < 0.001 and p = 0.007) than stages 0/I. Post-operatively, 8-OHG values were similar to controls (p = 0.053). Preoperatively, UA values (mg/dl) were significantly lower (p = 0.001), while postoperatively were similar to controls (p = 0.069). Oxidative RNA damage occurs in CRC patients. Stages II/III are associated with higher values of 8-OHG than stages 0/I. 8-OHG could act as a marker for the identification of patients with advanced disease. [ABSTRACT FROM AUTHOR]

Numerous reports find that Cd induces formation of reactive oxygen species (ROS) in plants. However, a general ROS pool is usually studied, without distinction of their production site. In the present study, we applied a mitochondria-specific antioxidant, MitoTEMPO, to elucidate the role of mitochondria-derived ROS in the response of soybean seedlings to short-term (48 h) Cd stress. The obtained results showed that Cd caused a reduction in root length and fresh weight and increase in the level of superoxide anion, hydrogen peroxide, markers of lipid peroxidation (thiobarbituric reactive substances, TBARS) and markers of RNA oxidation (8-hydroxyguanosine, 8-OHG) in seedling roots. Application of MitoTEMPO affected Cd uptake in a dose-dependent manner and diminished the Cd-dependent induction of superoxide anion and lipid peroxidation.

Abstract Introduction Oxidative stress leads to many kinds of diseases. Currently, urinary 8-hydroxydeoxyguanosine (8-OHdG) is widely measured as an oxidative stress biomarker. There is a specific advantage if saliva can be used as the sample to measure the oxidative stress biomarker, because saliva is much easier to collect than urine. In this study, we investigated the measurement of 8-hydroxyguanine (8-OHGua) as an oxidative stress marker in saliva, by a column switching HPLC system equipped with an electrochemical detector (HPLC-ECD). Findings The 8-OHGua in saliva could be detected as a single peak by HPLC-ECD. The average level of 8-OHGua in saliva was 3.80 ng/mL in ordinary, non-smoking subjects. The salivary 8-OHGua levels of smokers were significantly higher than those of non-smokers. Conclusions Salivary 8-OHGua may be a useful noninvasive and promising oxidative stress biomarker.

The paper presents the findings of an optimization and validation results of electrochemical detection of 8-hydroxyguanine, a major marker of oxidative stress, using disposable screen-printed carbon electrode (SPCE) modified with carboxy-functionalized multi-walled carbon nanotubes (MWCNTs-COOH). Several types of bare and modified SPCEs were tested and modification with MWCNTs-COOH was found superior in terms of measured current response, level of background currents, and sensitivity in oxidation of 8-hydroxyguanine. The effect of pH and composition of supporting electrolyte on current response and peak potential were also evaluated. Validation parameters were calculated for optimum pH 7 of 0.01 mol dm−3 phosphate buffer. The sensor showed the highest sensitivity of 0.0625 µA dm3 μmol−1 within the linear range 0.3–12 μmol dm−3 with a limit of detection as low as 0.57 μmol dm−3. Extended linear range 0.3–240 μmol dm−3 is feasible with a sensitivity of 0.0523 µA dm3 μmol−1. Intra-day and inter-day assays within 3 days were performed to verify the repeatability of modified screen-printed carbon sensors. Finally, the sensor was utilized for electrochemical monitoring of stability of 8-hydroxyguanine when stored in room temperature, in the refrigerator, or frozen and thawed. [ABSTRACT FROM AUTHOR]

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (G O , 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the G O -induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by G O .

Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (HdhQ111). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of HdhQ111 cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals.

Guanine oxidation occurs in both DNA and the cellular nucleotide pool, and one of the major products is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). The mutagenic potentials of this oxidized base have been examined in various experimental systems. In this review, we summarize the mutagenicity of the base in mammalian cells. We also describe the effects of specialized DNA polymerases, DNA repair proteins, and nucleotide pool sanitization enzymes. [ABSTRACT FROM AUTHOR]

ABSTRACT Biallelic germline mutations of MUTYH, the gene encoding DNA glycosylase, cause MUTYH-associated polyposis (MAP), characterized by multiple colorectal adenomas and carcinoma(s). However, a considerable number of MUTYH variants are still functionally uncharacterized. Herein, we report the results of functional evaluation of nine missense-type MUTYH variant proteins in the Japanese population. The DNA glycosylase activity and ability to suppress mutations caused by 8-hydroxyguanine, an oxidized form of guanine, were examined for the nine variants of type 2 MUTYH, a nuclear form of the enzyme, by DNA cleavage activity assay and supF forward mutation assay, respectively. Both activities were severely defective in the p.N210S MUTYH type 2 variant corresponding to p.N238S in the reference MUTYH form and partially defective in p.R219G variant corresponding to p.R247G, but nearly fully retained in seven other variants examined. Our results suggest that p.N238S and p.R247G are likely to be pathogenic alleles for MAP. [ABSTRACT FROM AUTHOR]

Oxidative stress is described as disturbed oxidative balance between increased reactive oxygen species such as hydroxyl radical (OH·), superoxide radical (O2-), hydrogen peroxide (H2O2) which occurs during normal cellular metabolism and decreased antioxidants which have scavenging effects on free radicals. Reactive oxygen species resulted from increased oxidative stress attacks on to the double bonds of lipids, proteins and DNA bases, removing one hydrogen atom from structure and initiate to oxidative chain reactions. This process leads to cellular damage and death damaging intracellular macromolecules such as lipids, proteins and DNA. The damage of free radicals is detected measuring oxidative products such as malondialdehyde (MDA), protein carbonyl (PCO) and 8-hydroxyguanine derivatives (8-OHG, 8-hidroksi-2'- deoksiguanozin) in body fluids and various tissues. Although considerable amount of studies in this field, the effects of oxidative stress on cellular structures still remain unknown. In this review, we aimed to evaluate the biochemical aspects of oxidative stress, antioxidant systems and their mechanism of actions and oxidative products. [ABSTRACT FROM AUTHOR]

Introduction: In DNA, 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most pivotal oxidatively damaged bases and induces G:C→T:A transversion mutations. DNA polymerase λ preferentially inserts dCTP opposite GO in vitro, and this error-free bypass function is considered to be important after A base removal from GO:A pairs by the MUTYH DNA glycosylase. To examine the effects of reduced levels of DNA polymerase λ on the GO-induced mutations, the polymerase was knocked-down in human U2OS cells, and a shuttle plasmid DNA containing a GO:C pair at position 122 in the supF gene was transfected into the cells. The plasmid DNA replicated in the cells was introduced into an Escherichia coli indicator strain, to measure the supF mutant frequency. Results: The knockdown of DNA polymerase λ significantly enhanced the mutant frequency of the GO plasmid DNA. Contrary to our expectations, the knockdown did not promote the targeted G:C→T:A transversion. Instead, substitution mutations at G:C sites other than position 122 were enhanced in the cells. Conclusions: These results suggested that the knockdown of DNA polymerase λ induced action-at-a-distance mutagenesis in human cells when the GO:C pair was present in the DNA. [ABSTRACT FROM AUTHOR]

The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine has been widely studied as a marker of DNA oxidation; however, data on the occurrence of other metabolites in plasma that are related to DNA damage are scarce. We have applied an improved, sensitive, robust, and reliable method, involving solid phase extraction and ultrahigh-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS), to the precise quantitation of seven metabolites in the plasma of 15 elite triathletes after a 2-week training program. All compounds were eluted in the first 1.6 min, with limits of detection and quantification ranging between 0.001 and 0.3 ng.mL−1 and 0.009 and 0.6 ng.mL−1, respectively. Four compounds were detected in plasma: guanosine-3'-5'-cyclic monophosphate, 8-hydroxyguanine, 8-hydroxy-2'-deoxyguanosine, and 8-nitroguanosine. After two weeks of training, 8-hydroxyguanine exhibited the highest increase (from 0.031 ± 0.008 nM to 0.036 ± 0.012 nM) ( p < 0.05), which could be related to the enhanced activity of DNA-repairing enzymes that excise this oxidized base. Increased levels of guanosine-3'-5'-cyclic monophosphate and 8-hydroxy-2'-deoxyguanosine were also observed. In contrast, levels of 8-nitroguanosine ( p < 0.05) were significantly reduced, which might be a protective measure as this compound strongly stimulates the generation of superoxide radicals, and its excess is related to pathologies such as microbial (viral) infections and other inflammatory and degenerative disorders. The results obtained indicate an induced adaptive response to the increased oxidative stress related to elite training, and point to the benefits associated with regular exercise. [ABSTRACT FROM AUTHOR]

Pulsed discharge is used for sterilization and disinfection, but the details of the molecular mechanisms remain largely unknown. Since pulsed discharge generates reactive oxygen species (ROS), we analyzed the oxidative DNA damages after pulsed discharge treatment to consider the involvement of ROS in the damaging process. We applied pulsed discharge with cavitation to plasmid DNA molecules and estimated the yields of the damages by agarose gel electrophoresis. The treated DNA contained various oxidative DNA damages, including single and double strand breaks and base lesions. The yields of the damages increased in response to the energy used for pulsed discharge. We also measured the yield of 8-hydroxyguanine (8-OH-G), one of the major oxidative base lesions, in the treated plasmid DNA by mass spectrometry quantitatively and found that the yield of the oxidative base lesion corresponded to the increment of the applied energy. In addition, we observed the involvement of mutM gene, which is responsible for repair of 8-OH-G, in the increased sensitivity of Escherichia coli to pulsed discharge. Therefore, ROS seem to mediate the sterilization ability of pulsed discharge. [ABSTRACT FROM AUTHOR]

An electrochemical study of the oxidation of 8-hydroxyguanine (8-OH-Gua) at gold nanoparticles attached to single walled carbon nanotube modified edge plane pyrolytic graphite electrode (AuNP-SWCNT/EPPGE) has been carried out to develop a method for the self diagnosis of diabetes. The level of 8-OH-Gua, an important biomarker of oxidative DNA damage, is higher in urine of diabetic patients than control subjects. A detailed comparison has been made between the square wave voltammetric (SWV) response of SWCNT/EPPGE and AuNP-SWCNT/EPPGE towards the oxidation of 8-OH-Gua in respect of several essential analytical parameters viz. sensitivity, detection limit, peak current and peak potential. The AuNP-SWCNT/EPPGE exhibited a well defined anodic peak at potential of ~ 221 mV for the oxidation of 8-OH-Gua as compared to ~ 312 mV using SWCNT/EPPGE at pH = 7.2. Under optimized conditions linear calibration curve for 8-OH-Gua is obtained over a concentration range of 0.01–10.0 nM in phosphate buffer solution (PBS) of pH = 7.2 with detection limit and sensitivity of 5.0 (± 0.1) pM and 4.9 (± 0.1) μA nM − 1 , respectively. The oxidation of 8-OH-Gua occurred in a pH dependent process and the electrode reaction followed adsorption controlled pathway. The electrode exhibited an efficient catalytic response with good reproducibility and stability. The method has been found selective and successfully implemented for the determination of 8-OH-Gua in urine samples of diabetic patients. [ABSTRACT FROM AUTHOR]

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine, G°) is a major oxidized base that is considered to play pivotal roles in the pathogenesis of various diseases, including cancer. G° induces G:C → T:A transversions at the damage site and untargeted (action-at-a-distance) mutations of G bases at 5′-GpA sequences. In this study, we examined the distribution of the action-at-a-distance mutations and the effects of the replication origin position relative to G° on the untargeted mutagenesis. The G° base was introduced into two shuttle plasmids, each with the SV40 replication origin at a different position with respect to the supF gene. The oxidized base was located at an upstream or downstream site (outside of the gene), or the center of the region encoding the pre-tRNA sequence of the gene, in the sense strand. These shuttle plasmids were introduced into human U2OS cells. The action-at-a-distance mutations were more frequently induced when the G° base was located downstream of the supF gene than upstream of the gene. In addition, more action-at-a-distance mutations were observed when the SV40 origin was present on the 5′-side of the G° base. These results indicated that the action-at-a-distance mutations are predominantly induced on the 5′-side of the lesion and occurred more frequently when the damaged base was located on the lagging strand template. • 8-Oxo-7,8-dihydroguanine induces untargeted base substitution mutations. • The untargeted mutations are primarily on the 5′-side of the oxidized base. • The oxidized base on the lagging strand template more frequently induces mutations. • Positions of the damaged base and the replication origin influence the mutagenesis. [ABSTRACT FROM AUTHOR]

DNA polymerase λ preferentially inserts dCTP opposite 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) in vitro, and this function is considered to be important after A base removal from GO:A pairs by the MUTYH DNA glycosylase. However, dCTP incorporation would promote A:T→C:G transversion mutations induced by 8-oxo-7,8-dihydrodGTP (dGOTP), the mutation triggered by the incorporation of dGOTP opposite A in the template DNA. In this study, double-stranded plasmid DNA containing the GO: A pair, an intermediate in the dGO TP mutagenesis pathway, was transfected into human cells in which DNA polymerase λ was knocked down. The knockdown of DNA polymerase λ significantly reduced the frequency of A:T→C:G transversion mutations induced by the GO:A pair although the knockdown effect was small. These results suggested that DNA polymerase λ is involved in the mutagenesis processes of GO, generated by dGOTP, in the nucleotide pool. [ABSTRACT FROM AUTHOR]

Hypercholesterolemia is a major risk factor for atherosclerosis and related occlusive vascular diseases. We investigated the effect of low-dose fluvastatin (2 mg kg−1 day−1) on antioxidant enzyme activities [superoxide dismutase (SOD), catalase], vascular reactivity changes and oxidatively induced DNA damage in early stage of atherosclerosis in hypercholesterolemic rabbits. The animals were divided into three groups each composed of 10 rabbits. The control group received a regular rabbit chow diet, and the cholesterol group had hypercholesterolemic diet (2%, 4 weeks). The fluvastatin group was given hypercholesterolemic diet plus fluvastatin. Dietary intake of cholesterol significantly increased total cholesterol levels in rabbits (control, 0.85 ± 0.29; cholesterol, 12.04 ± 4.61; fluvastatin, 8.07 ± 2.72 mmol l−1 ). Hypercholesterolemic diet revealed discernible fatty streaks in arcus aortae. Fluvastatin significantly reduced the areas of the lesions. The diet significantly increased SOD activities in both erythrocyte and tissue. Treatment with fluvastatin normalized the increased activity of SOD in both erythrocyte and aortic tissues from the cholesterol group. Cholesterol feeding decreased the sensitivity to acetylcholine, and treatment with fluvastatin significantly restored the diminished sensitivity to acetylcholine in thoracic aortae. Cholesterol feeding caused oxidatively induced DNA damage in liver tissues determined by the increased levels of 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Fluvastatin decreased only FapyGua level in liver. In conclusion, our results may suggest that fluvastatin seems to play a protective role on high cholesterol-induced oxidative stress and DNA damage. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: MUTYH, a human ortholog of MutY, is a post-replicative DNA glycosylase, highly conserved throughout evolution, involved in the correction of mismatches resulting from a faulty replication of the oxidized base 8-hydroxyguanine (8-oxodG). In particular removal of adenine from A:8-oxodG mispairs by MUTYH activity is followed by error-free base excision repair (BER) events, leading to the formation of C:8-oxodG base pairs. These are the substrate of another BER enzyme, the OGG1 DNA glycosylase, which then removes 8-oxodG from DNA. Thus the combined action of OGG1 and MUTYH prevents oxidative damage-induced mutations, i.e. GC->TA transversions. Germline mutations in MUTYH are associated with a recessively heritable colorectal polyposis, now referred to as MUTYH-associated polyposis (MAP). Here we will review the phenotype(s) associated with MUTYH inactivation from bacteria to mammals, the structure of the MUTYH protein, the molecular mechanisms of its enzymatic activity and the functional characterization of MUTYH variants. The relevance of these results will be discussed to define the role of specific human mutations in colorectal cancer risk together with the possible role of MUTYH inactivation in sporadic cancer. [Copyright &y& Elsevier]

Abstract: Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (HdhQ111). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of HdhQ111 cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals. [Copyright &y& Elsevier]

The study of Alzheimer's disease neuropathology has been intimately associated with the field of oxidative stress for nearly 20 years. Indeed, increased markers of oxidative stress have been associated with this neurodegenerative condition, resulting from oxidation of lipids, proteins and nucleic acids. Increased nuclear and mitochondrial DNA oxidation are observed in Alzheimer's disease, stemming from increased reactive oxygen species attack to DNA bases and from the impairment of DNA repair mechanisms. Moreover, mitochondrial DNA is found to be more extensively oxidized than nuclear DNA. This review is intended to summarizes the most important cellular reactive oxygen species producers and how mitochondrial dysfunction, redox-active metals dyshomeostasis and NADPH oxidases contribute to increased oxidative stress in Alzheimer's disease. A summary of the antioxidant system malfunction will also be provided. Moreover, we will highlight the mechanisms of DNA oxidation and repair. Importantly, we will discuss evidence relating the DNA repair machinery and accumulated DNA oxidation with Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Abstract: 8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-hydroxy-2′- deoxyguanosine 5′-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H2O2, and the induced mutations were analyzed. Moreover, the 2′-deoxyriboside 5′-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H2O2 treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2′-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2′-deoxyriboside 5′-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA. [Copyright &y& Elsevier]

Purpose: The aim of this work was to study the combined effect of sodium and acetate ions on the radiation yield of 8-hydroxyguanine (8-OHG), one of the major DNA base lesions induced by free radicals. Materials and methods: Aqueous solutions of DNA and 2′-deoxyguanosine 5′-monophosphate (dGMP) with various concentrations of sodium acetate and sodium perchlorate were γ-irradiated, enzymatically digested and analyzed by high-performance liquid chromatography (HPLC) methods. Results: It was found that both salts decrease the 8-OHG radiation yield in the concentration range studied for both DNA and dGMP, except in the case of dGMP wherein an increase in yield occurs in the concentration range from 0.1-1 mM. The dependence of the 8-hydroxy-2′-deoxyguanosine radiation yield on the concentration of both sodium acetate and sodium perchlorate have different shapes and have steeper slopes for the DNA compared with the dGMP solutions. Conclusions: The observed decrease in the radiation yield of 8-OHG with increasing concentrations of sodium acetate is consistent with the hypothesis that sodium acetate produces two concentration-dependent effects in the DNA solutions: (1) A conformational change in the DNA caused by Na++ counterions; and (2) free radical reactions related to the radiolysis of acetate ion. [ABSTRACT FROM AUTHOR]

Abstract: A simple and reliable method based on voltammetry is proposed for the determination of oxidative DNA damage by the simultaneous determination of guanine and 8-hydroxygunine using single-walled carbon nanotubes modified edge plane pyrolytic graphite electrode (SWNT/EPPGE). In acid-hydrolyzed calf thymus DNA, two well-defined peaks at ∼312 and ∼502mV corresponding to the oxidation of 8-hydroxyguanine and guanine, respectively in phosphate buffer solution (PBS) of pH 7.2 were observed in square wave voltammetry. Remarkable enhancement in the oxidation peak current of both the compounds was observed along with the negative shift of peak potentials using SWNT/EPPGE as compared to bare EPPGE. The detection limits of guanine and 8-hydroxyguanine were calculated to be 0.05×10−9 and 0.01×10−9 molL−1, respectively. The limits of quantification were found as 0.17×10−9 and 0.34×10−10 molL−1 for guanine and 8-hydroxyguanine, respectively. [ABSTRACT FROM AUTHOR]

Abstract: 8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2′-deoxyguanosine 5′-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C→T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T→C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T→C:G mutations caused by 8-oxo-dGTP. [Copyright &y& Elsevier]

Abstract: The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C→T:A and A:T→C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases η and ζ by siRNAs enhanced the G:C→T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases η and ζ, suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase η slightly decreased the A:T→C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases η and ζ are involved in the bypass of 8-OH-Gua in human cells. [Copyright &y& Elsevier]

Abstract: Alcohol consumption is known to have opposing effects on carcinogenesis: promotion and prevention. In this study, we examined the effects of 12% ethanol on oxidative DNA damage accumulation and its repair in mouse livers treated with 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), a well-known hepatic carcinogen. We previously reported that 3′-MeDAB increased 8-hydroxyguanine (8-OH-Gua) accumulation and its repair activity, accompanied by the fragmentation of 8-oxoguanine DNA glycosylase 1 (OGG1), the main repair enzyme of 8-OH-Gua. The present results showed that 12% ethanol intake attenuated the 8-OH-Gua accumulation, but not the fragmentation of OGG1 induced by 3′-MeDAB. Additionally, no significant changes in oxidative status, as monitored by lipid peroxidation (LPO), were observed among the 3′-MeDAB-treated mouse livers with/without alcohol administration. These findings suggested that 12% ethanol consumption may reduce the risk of 3′-MeDAB-induced carcinogenesis by decreasing 8-OH-Gua accumulation. [Copyright &y& Elsevier]

Oxidatively damaged bases induce mutations and are involved in cancer initiation. 8-Oxo-7,8-dihydroguanine (G°, 8-hydroxyguanine) is an abundant oxidized base that induces targeted G:C→T:A transversions in human cells, as well as untargeted base substitution (action-at-a-distance) mutations of the G bases of 5′-GpA-3′ dinucleotides. The action-at-a-distance mutations become more frequent than the targeted transversions when the amount of Werner syndrome (WRN) protein is decreased. In this study, OGG1, the major DNA glycosylase for the damaged base, and WRN were knocked down in isolation and in combination in human U2OS cells, and a shuttle plasmid carrying G° was introduced into the knockdown cells. Interestingly, fewer action-at-a-distance mutations were observed in the WRN plus OGG1 double knockdown cells, as compared to the WRN single knockdown cells. These results indicated the paradoxical role of OGG1, as an accelerator of the action-at-a-distance mutations by the oxidized guanine base. • 8-Oxo-7,8-dihydroguanine induces untargeted base substitution mutations. • OGG1 is the major DNA glycosylase for the oxidized base. • Knockdown of Werner syndrome (WRN) protein enhances the untargeted mutations. • OGG1 knockdown suppresses the increase in the mutations by the WRN reduction. • The paradoxical role of OGG1 in the untargeted mutations is indicated. [ABSTRACT FROM AUTHOR]

Abstract: Elevated levels of oxidatively induced DNA lesions have been reported in malignant pancreatic tissues relative to normal pancreatic tissues. However, the ability of the pancreatic cancer cells to remove these lesions has not previously been addressed. This study analyzed the effectiveness of the pancreatic cancer cell line, BxPC-3 to repair 8-hydroxyguanine (8-OH-Gua) relative to a nonmalignant cell line. We show that BxPC-3 cells repair 8-OH-Gua less effectively than the nonmalignant cells. This repair deficiency correlated with significant downregulation of the hOGG1 protein and the corresponding mRNA (30-fold lower than GAPDH) in BxPC-3 cell line. The repair defect was complemented in vivo by transient transfection of the hOGG1 gene and in vivo by recombinant hOGG1. These results are the first to show a deficiency of 8-OH-Gua repair in BxPC-3 cells, implicating this defect in the risk factor of pancreatic cancer. [Copyright &y& Elsevier]

Purpose: To detect the genotoxic effects of extremely low frequency (ELF) -magnetic fields (MF) on oxidative DNA base modifications [8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde)] in rat leucocytes, measured following exposure to ELF-MF. Materials and methods: After exposure to ELF-MF (50 Hz, 100 and 500 μT, for 2 hours/day during 10 months), DNA was extracted, and measurement of DNA lesions was achieved by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). Results: Levels of FapyAde, FapyGua and 8OHdG in DNA were increased by both 100 μT and 500 μT ELF-MF as compared to a cage-control and a sham group; however, statistical significance was observed only in the group exposed to 100 μT. Conclusion: This is the first study to report that ELF-MF exposure generates oxidatively induced DNA base modifications which are mutagenic in mammalian cells, such as FapyGua, FapyAde and 8-OH-Gua, in vivo. This may explain previous studies showing DNA damage and genomic instability. These findings support the hypothesis that chronic exposure to 50-Hz MF may be potentially genotoxic. However, the intensity of ELF-MF has an important influence on the extent of DNA damage. [ABSTRACT FROM AUTHOR]

Ionizing radiation-induced formation of genomic DNA damage can be modulated by nearby chemical species such as heavy metal ions, which can lead to non-linear dose response. To investigate this phenomenon, we studied cell survival and formation of 8-hydroxyguanine (8-OHG) base modifications and double strand breaks (DSB) caused by combined action of cadmium (Cd) and gamma radiation in cultured medaka fish (Oryzias latipes) fibroblast cells. Our data show that the introduction of Cd leads to a significant decrease in the fraction of surviving cells and to increased sensitivity of cells to ionizing radiation (IR). Cd also appears to cause non-linear increases in radiation-induced yields of 8-OHG and DSB as dose-yield plots of these lesions exhibit non-linear S-shaped curves with a sharp increase in the yields of lesions in the 10-20 μM range of Cd concentrations. The combined action of ionizing radiation and Cd leads to increased DNA damage formation compared to the effects of the individual stressors. These results are consistent with a hypothesis that the presence of Cd modulates the efficiency of DNA repair systems thus causing increases in radiation-induced DNA damage formation and decreases in cell survival. [ABSTRACT FROM AUTHOR]

Abstract: Findings from epidemiologic studies have been important in evaluating risk of exposure to many contaminants in drinking water. In the case of bromate, a byproduct of ozone disinfection of water, it is unlikely that observational studies of populations exposed to bromate in drinking water will be as revealing as studies of other contaminants, unless risks are much higher than predicted from laboratory studies of rodents. Occupational exposure to bromate has occurred in the flour milling and baking industries, as well as in chemical production of potassium bromate, used as a flour additive. The feasibility of a cohort study of bromate-exposed workers should be evaluated by studying the conditions and levels of exposure in these occupational settings. Bromate exposure causes oxidative damage to guanine bases of DNA, producing 8-hydroxy-guanine (8-OH-Gua), which is excised by 8-oxoguanosine glycosylase (OGG1) and excreted in the urine. Polymorphic variants of OGG1 in human populations have been associated with elevated cancer risk. 8-OH-Gua and 8-hydroxy-deoxyguanosine (8-OHdG) have been used as biomarkers of oxidative damage in many human studies, and it would be feasible to employ these indicators in controlled clinical experimental settings to see if exposure to bromate in water at levels close to the maximum contaminant level influences urinary levels of excretion, and if so, to help quantify the level of oxidative damage. Such a study could fill an important data gap by providing human data to help estimate the carcinogenic risk from this exposure. [Copyright &y& Elsevier]

Increasing evidence suggests that oxidative damage is associated with normal aging and several neurodegenerative diseases. Mild cognitive impairment (MCI), the phase between normal aging and early dementia, is a common problem in the elderly with many subjects going on to develop Alzheimer's disease (AD). Although increased DNA oxidation is observed in the AD brain, it is unclear when the oxidative damage begins. To determine if DNA oxidation occurs in the brain of subjects with MCI, we quantified multiple oxidized bases in nuclear and mitochondrial DNA isolated from frontal, parietal and temporal lobes and cerebellum of short post-mortem interval autopsies of eight amnestic patients with MCI and six age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring. We found statistically significant elevations ( p < 0.05) of 8-hydroxyguanine, a widely studied biomarker of DNA damage, in MCI nuclear DNA from frontal and temporal lobe and in mitochondrial DNA from the temporal lobe compared with age-matched control subjects. Levels of 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine were significantly elevated in nuclear DNA from all three neocortical regions in MCI. Statistically significant elevations of 4,6-diamino-5-formamidopyrimidine were also observed in mitochondrial DNA of MCI temporal, frontal and parietal lobes. These results suggest that oxidative damage to nuclear and mitochondrial DNA occurs in the earliest detectable phase of AD and may play a meaningful role in the pathogenesis of this disease. [ABSTRACT FROM AUTHOR]

Abstract: Chromosome DNA is continuously exposed to various endogenous and exogenous mutagens. Among them, oxidation is one of the most common threats to genetic stability, and multiple DNA repair enzymes protect chromosome DNA from the oxidative damage. In Escherichia coli, three repair enzymes synergistically reduce the mutagenicity of oxidized base 8-hydroxy-guanine (8-OH-G). MutM DNA glycosylase excises 8-OH-G from 8-OH-G:C pairs in DNA and MutY DNA glycosylase removes adenine incorporated opposite template 8-OH-G during DNA replication. MutT hydrolyzes 8-OH-dGTP to 8-OH-dGMP in dNTP pool, thereby reducing the chance of misincorporation of 8-OH-dGTP by DNA polymerases. Simultaneous inactivation of MutM and MutY dramatically increases the frequency of spontaneous G:C to T:A mutations, and the deficiency of MutT leads to the enhancement of T:A to G:C transversions more than 1000-fold over the control level. In humans, the functional homologues of MutM, MutY and MutT, i.e., OGG1, MUTYH (MYH) and MTH1, contribute to the protection of genomic DNA from oxidative stress. Interestingly, several polymorphic forms of these proteins exist in human populations, and some of them are suggested to be associated with cancer susceptibility. Here, we review the polymorphic forms of OGG1, MUTYH and MTH1 involved in repair of 8-OH-G and 8-OH-dGTP, and discuss the significance of the polymorphisms in the maintenance of genomic integrity. We also summarize the polymorphic forms of human DNA polymerase η, which may be involved in damage tolerance and mutagenesis induced by oxidative stress. [Copyright &y& Elsevier]

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD. [ABSTRACT FROM AUTHOR]

Substantial evidence suggest that oxidative damage may play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). We examined levels of 8-Hydroxy-2'-deoxyguanosine (8OH2'dG) in the nuclear DNA from the spinal cord, frontal cortex, striatum and cerebellum from G93A mice at 60, 90, and 120 days of age. We also used in vivo microdialysis to measure free levels of 8OH2'dG and 8-Hydroxyguanine (8OHG) at the same time points in the frontal cortex of G93A mice. Increased 8OH2'dG DNA levels were observed in the spinal cord (at 60, 90 and 120 days), in the cortex (at 90, and 120 days), and in the striatum (at 120 days), as compared to age-matched littermate controls. No significant changes were found in the cerebellum at any of the time points studied. Free levels of 8OH2'dG in the cortex of G93A mice were increased, as compared to control mice, at 90 and 120 days. Free levels of 8OHG were found to be significantly higher at 120 days of age in control mice than in G93A mice. These results provide evidence that in this model of ALS oixidative DNA-damage is increased and base excision-repair may be deficient. [ABSTRACT FROM AUTHOR]