Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

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 (GO, 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 GO-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 GO.

Role of 8-hydroxyguanine DNA glycosidase 1 deficiency in exacerbating diabetic cardiomyopathy through the regulation of insulin resistance.

Diabetic cardiomyopathy (DCM) is a heart failure syndrome, and is one of the major causes of morbidity and mortality in diabetes. DCM is mainly characterized by ventricular dilation, myocardial hypertrophy, myocardial fibrosis and cardiac dysfunction. Clinical studies have found that insulin resistance is an independent risk factor for DCM. However, its specific mechanism of DCM remains unclear. 8-hydroxyguanine DNA glycosylase 1(OGG1)is involved in DNA base repair and the regulation of inflammatory genes. In this study, we show that OGG1 was associated with the occurrence of DCM. for the first time. The expression of OGG1 was increased in the heart tissue of DCM mice, and OGG1 deficiency aggravated the cardiac dysfunction of DCM mice. Metabolomics show that OGG1 deficiency resulted in obstruction of glycolytic pathway. At the molecular level, OGG1 regulated glucose uptake and insulin resistance by interacting with PPAR-γ in vitro. In order to explore the protective effect of exogenous OGG1 on DCM, OGG1 adeno-associated virus was injected into DCM mice through tail vein in the middle stage of the disease. We found that the overexpression of OGG1 could improve cardiac dysfunction of DCM mice, indicating that OGG1 had a certain therapeutic effect on DCM. These results demonstrate that OGG1 is a new molecular target for the treatment of DCM and has certain clinical significance.

Action-at-a-distance mutations induced by 8-oxo-7,8-dihydroguanine are dependent on APOBEC3.

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 5'-GpA-3' dinucleotides (action-at-a-distance mutations). The 5'-GpA-3' sequences are complementary to the 5'-TpC-3' 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.

Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

Chemotherapy resistance is the dominant challenge in the treatment of acute myeloid leukemia (AML). Nuclear factor E2-related factor 2 (Nrf2) exerts a vital function in drug resistance of many tumors. Nevertheless, the potential molecular mechanism of Nrf2 regulating the base excision repair pathway that mediates AML chemotherapy resistance remains unclear. Here, in clinical samples, we found that the high expression of Nrf2 and base excision repair pathway gene encoding 8-hydroxyguanine DNA glycosidase (OGG1) was associated with AML disease progression. In vitro, Nrf2 and OGG1 were highly expressed in drug-resistant leukemia cells. Upregulation of Nrf2 in leukemia cells by lentivirus transfection could decrease the sensitivity of leukemia cells to cytarabine, whereas downregulation of Nrf2 in drug-resistant cells could enhance leukemia cell chemosensitivity. Meanwhile, we found that Nrf2 could positively regulate OGG1 expression in leukemia cells. Our chromatin immunoprecipitation assay revealed that Nrf2 could bind to the promoter of OGG1. Furthermore, the use of OGG1 inhibitor TH5487 could partially reverse the inhibitory effect of upregulated Nrf2 on leukemia cell apoptosis. In vivo, downregulation of Nrf2 could increase the sensitivity of leukemia cell to cytarabine and decrease OGG1 expression. Mechanistically, Nrf2-OGG1 axis-mediated AML resistance might be achieved by activating the AKT signaling pathway to regulate downstream apoptotic proteins. Thus, this study reveals a novel mechanism of Nrf2-promoting drug resistance in leukemia, which may provide a potential therapeutic target for the treatment of drug-resistant/refractory leukemia.

PlexinA1-deficient mice exhibit decreased cell density and augmented oxidative stress in parvalbumin-expressing interneurons in the medial prefrontal cortex.

PlexinA1 (PlxnA1) is a transmembrane receptor for semaphorins (Semas), a large family of axonal guidance cues vital during neural development. PlxnA1 is expressed in embryonic interneurons, and PlxnA1 deletion in mice leads to less interneurons in the developing cortex. In addition, PlxnA1 has been identified as a schizophrenia susceptibility gene. In our previous study, PlxnA1 knockout (KO) mice under a BALB/cAJ genetic background exhibited significantly increased self-grooming and reduced prepulse inhibition, a reliable phenotype for investigating the neurobiology of schizophrenia. However, the mechanism underlying the abnormal behavior of PlxnA1 KO mice remains unclear. We first confirmed PlxnA1 mRNA expression in parvalbumin-expressing interneurons (PV cells) in the medial prefrontal cortex (mPFC) of adult mice. Immunohistochemical analysis (IHC) showed significantly decreased densities of both GABAergic neurons and PV cells in the mPFC of PlxnA1 KO mice compared with wild type mice (WT). PV cells were found to express molecule interacting with CasL 1 (MICAL1), an effector involved in Sema-Plxn signaling for axon guidance, suggesting MICAL1 and PlxnA1 co-expression in PV cells. Furthermore, IHC analysis of 8-oxo-dG, an oxidative stress marker, revealed significantly increased oxidative stress in PlxnA1-deficient PV cells compared with WT. Thus, increased oxidative stress and decreased PV cell density in the mPFC may determine the onset of PlxnA1 KO mice's abnormal behavior. Accordingly, deficient PlxnA1-mediated signaling may increase oxidative stress in PV cells, thereby disrupting PV-cell networks in the mPFC and causing abnormal behavior related to neuropsychiatric diseases.

Mitochondria Specific Antioxidant, MitoTEMPO, Modulates Cd Uptake and Oxidative Response of Soybean Seedlings.

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.

Biased distribution of action-at-a-distance mutations by 8-oxo-7,8-dihydroguanine.

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.

A Pilot Study on the Association of Lead, 8-Hydroxyguanine, and Malondialdehyde Levels in Opium Addicts' Blood Serum with Illicit Drug Use and Non-Addict Persons.

While a large body of literature has shown the health problems of illicit drug use, research is needed on how substance abuse impacts DNA damage and contaminants in blood, especially given Pb-contaminated opium. This pilot study aimed to evaluate the levels of lead (Pb), 8-hydroxy di-guanine (8-oxo-Gua), and malondialdehyde (MDA) in the blood serum of opium addicts and non-addict people. The current study is a case-control study with a cross-sectional design. A sample of 50 opium-addicted and non-addict adults were chosen for this study using convenience and random sampling methods. Participants were divided into two groups: addicts and non-addicts. The atomic absorption spectroscopy method was used to measure the quantity of Pb, and the Enzyme-Linked Immunosorbent Assay (ELISA) method was used to measure the amount of 8-oxo-Gua and MDA. The data were analyzed using an independent t-test. The results show that the amount of Pb in the blood serum of addicted women and men was higher than levels in non-addict men and women, for the study participants (p-value = 0.001). Blood levels were not significantly different between addicts and non-addicts for men or women for 8-oxo-Gua (p-value = 0.647 for women and p-value = 0.785 for men) and MDA (p-value = 0.867 for women and p-value = 0.995 for men). In general, addicts' blood Pb levels were found to be substantially higher than those of normal non-addict persons in this pilot study. As a result, testing for blood Pb levels in addicts may be informative in instances when symptoms are inconclusive.

Paradoxical role of the major DNA repair protein, OGG1, in action-at-a-distance mutation induction by 8-oxo-7,8-dihydroguanine.

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.

Urine 8-Hydroxyguanine (8-OHG) in Patients Undergoing Surgery for Colorectal Cancer.

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 < 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). CONCLUSION: 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.

Salivary 8-hydroxyguanine as a lifestyle-related oxidative stress biomarker in workers.

Oxidative stress is a risk factor for lifestyle-related diseases, such as cancer. Investigations of the factors that increase or decrease oxidative stress contribute to disease prevention. In the present study, we focused on the 8-hydroxyguanine (8-OHGua) in saliva, as a new oxidative stress biomarker. The relationship between lifestyles and salivary 8-OHGua levels in 541 Japanese subjects was analyzed. The salivary 8-OHGua levels were significantly elevated in older persons, as well as those who smoke, have hypertension, or excess visceral fat. By contrast, statistically significant lower levels of 8-OHGua were observed in persons who moderately exercised or recently drank green tea or coffee. The direct collection of saliva, without any special collecting device, was suitable for the 8-OHGua analysis. The present results suggest that oxidative stress can be measured in a non-invasive manner with easily collectable saliva, and the salivary 8-OHGua may be a useful biomarker for lifestyle-related disease prevention.

Facile synthesis of copper ferrite nanoparticles with chitosan composite for high-performance electrochemical sensor.

Herein, the synthesis of copper ferrite nanoparticles (CuFe2O4 NPs)/chitosan have been prepared by sonochemical route under ultrasonic irradiation bath at 40 kHz and 50 W. A high sensitive and stable modified electrochemical sensor was developed using a composition of copper ferrite nanoparticles coordinated with biopolymer through a facile ultrasound approach. Besides, power and frequency parameters are highly important for sonochemical synthesis and specifically structure, and size of the nanomaterials development during the ultrasonic irradiation time. In this work, ultrasonic bath was used to synthesis of CuFe2O4 nanomaterial at 40 kHz with 1 h. CuFe2O4/chitosan was characterized by FESEM, EDX, XRD and electrochemical methods. Furthermore, 8-hydroxyguanine is one of biomarker by oxidative stress. The concentrations of 8-hydroxyguanine within a cell are a measurement of oxidative stress in human body. Consequently, the measurement of 8-hydroxyguanine in blood serum samples with high specificity is of greatest importance. The CuFe2O4/chitosan modified electrode is displayed a low detection limit of 8.6 nM and long linear range (0.025-697.175 µM).

Diurnal variation of salivary oxidative stress marker 8-hydroxyguanine.

INTRODUCTION: Oxidative stress is a risk factor for life-style related diseases, including cancer. We recently reported that the oxidative stress marker 8-hydroxyguanine (8-OHGua) can be measured in saliva non-invasively. Understanding the diurnal pattern of salivary 8-OHGua levels is crucial for evaluating the oxidative stress. In this study, we analyzed the diurnal variation of salivary 8-OHGua levels. FINDINGS: The salivary 8-OHGua levels were relatively stable in the daytime (10:00-22:00). The daytime 8-OHGua levels seemed to represent the individual oxidative stress status. The average amount and the variation of the salivary 8-OHGua levels immediately after awakening were higher than those of the daytime levels. CONCLUSIONS: The 8-OHGua levels in saliva exhibited diurnal variation. The levels were higher at the time of awakening. At this point, the daytime levels of salivary 8-OHGua may be appropriate for evaluating the individual oxidative stress status. Further study is needed for understanding and utilizing the 8-OHGua levels at the time of awakening.

Mechanistic processes of resveratrol in inhibiting the oxidative damage of guanine, as evidenced by UHPLC-MS2.

Resveratrol, as one of the stilbenoids, is present in abundance in wine grapes and has been shown to selectively quench 1O2. DNA is oxidized by 1O2 causing irreparable functional damage, and of the nucleic acids, guanine is the most susceptible. An agarose gel electrophoresis assay demonstrated that DNA was damaged by 1O2 with less than 5 min of UVA irradiation, and also that 5 mM resveratrol dissolved in MeOH could relieve the observed oxidation stress. Ultra-high performance liquid chromatography coupled with mass spectrometry was performed to reveal the mechanism. Four guanine oxidation products at m/z 140.0334 [M-H]-(1), DGh, 8-oxoG, Sp and two conjugates at m/z 377.1104 [M-H]- and 391.0907 [M-H]- were identified and quantified. Thus, we propose the mechanism that the phenol ring of resveratrol links with the free amino groups (NH) of guanine at the beginning of 1O2 attack to form m/z 377.1104 [M-H]-, however, as 1O2 is able to attack the amino groups continuously, resveratrol can efficiently react with 1O2 prior to damage, and form m/z 391.0907 [M-H]- thereby protecting guanine.

Effects of the chloro-s-triazine herbicide terbuthylazine on DNA integrity in human and mouse cells.

Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.

Measurement of 8-hydroxyguanine as an oxidative stress biomarker in saliva by HPLC-ECD.

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.

Mutations induced by 8-hydroxyguanine (8-oxo-7,8-dihydroguanine), a representative oxidized base, in mammalian cells.

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.

Reduced expression of the DNA glycosylase gene MUTYH is associated with an increased number of somatic mutations via a reduction in the DNA repair capacity in prostate adenocarcinoma.

8-Hydroxyguanine (8OHG), a major oxidative DNA lesion, is known to accumulate in prostate cancer; however, the status of one of its repair enzymes, MUTYH, in prostate cancer remains to be elucidated. In this study, we showed that the expression levels of MUTYH mRNA and protein were significantly lower in prostate cancer than in non-cancerous prostatic tissue by examining two independent, publicly available databases and by performing an immunohistochemical analysis of prostate cancer specimens obtained at our hospital, respectively. About two-thirds of the prostate cancers exhibited a reduced MUTYH expression. When the effect of reduced MUTYH expression in prostate adenocarcinoma on the somatic mutation load was examined using data from the Cancer Genome Atlas (TCGA) database, the numbers of total somatic mutations and somatic G:C to T:A mutations were significantly higher in the reduced MUTYH expression group than in the other group (P < 0.0001 and P = 0.0013, respectively). To determine the reason why reduced MUTYH expression leads to somatic mutation loads in prostate adenocarcinoma, we compared the DNA repair capacities between PC-3 prostatic cell line derived clones with different MUTYH expression levels. Both the capacities to cleave DNA containing adenine:8OHG mispairs and to suppress mutations caused by 8OHG were significantly lower in prostatic cell lines with lower MUTYH expression than in prostatic cell lines with higher MUTYH expression. These results suggested that reduced MUTYH expression is associated with somatic mutation loads via a reduction in DNA repair capacity in prostate adenocarcinoma. © 2016 Wiley Periodicals, Inc.

Functional Evaluation of Nine Missense-Type Variants of the Human DNA Glycosylase Enzyme MUTYH in the Japanese Population.

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.

Effect of elite physical exercise by triathletes on seven catabolites of DNA oxidation.

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.