Age-dependent increases in the oxidative damage of DNA, RNA, and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS: urinary 8-oxoguanosine as a novel biomarker of aging

A sensitive and accurate isotope-diluted LC-MS/MS method was developed for determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn), derived from DNA, and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), derived from RNA, in various tissue specimens obtained from normal SAMR1 and senescence-accelerated SAMP8 mice. An age-dependent accumulation of oxidative DNA and RNA damage was observed in all the organs examined, namely, the brain, liver, lungs, heart, kidneys, and testes. Among these, the brain samples exhibited the highest values for DNA damage. These age-related increases in the 8-oxoguanine content in DNA and RNA occurred more rapidly in SAMP8 than in SAMR1 mice. Age-related increases in the contents of 8-oxo-dGsn and 8-oxo-Gsn were also observed in the plasma and urine; however, the ratios of 8-oxo-Gsn to 8-oxo-dGsn in these samples were considerably higher (6 to 13) compared with the values for the samples derived from other tissues (roughly 1), indicating that measurement of 8-oxo-Gsn in urine could be a novel means of evaluating the aging process.     

Levels of 8-oxo-dGsn and 8-oxo-Gsn in random urine are consistent with 24 h urine in healthy subjects and patients with renal disease

Oxidatively generated damage to nucleic acids may play an important role in the pathophysiological processes of a variety of diseases. 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) are oxidatively generated products of DNA and RNA, respectively. Our previous studies have suggested that the amounts of 8-oxo-dGsn and 8-oxo-Gsn in urine were considerably higher than other body fluid or tissue. The aim of this study was to investigate whether 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples are consistent with those in 24?h urine samples in healthy subjects and patients with renal disease. A total of 16 healthy subjects and 104 renal disease patients were enrolled in this study, and their random and 24?h urine samples were collected. The levels of urinary 8-oxo-dGsn and 8-oxo-Gsn were quantified by LC-MS/MS and corrected by creatinine. Regardless of healthy subjects or renal disease patients, the levels of oxidised nucleosides in random urine samples were consistent with 24?h urine samples. Regardless of the age bracket, there is no significant difference between random samples and 24?h urine samples. In conclusion, 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples could replace those in 24?h urine samples, and were considered as the representative of the level of systemic oxidative stress for the whole day.     

Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats

Abstract

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p?≤?0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p?=?0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.     

Increased oxidative damage of RNA in liver injury caused by hepatitis B virus (HBV) infection

To evaluate the urinary levels of 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in liver injury patients with hepatitis B virus (HBV) infection and to explore the relationship between urinary 8-oxo-dGsn or 8-oxo-Gsn and degree of liver damage. We enrolled 138 liver injury patients with HBV infection and 169 age- and sex-matched healthy controls in this study. A sensitive and accurate isotope-diluted liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) method was used to measure the urinary levels of 8-oxo-Gsn and 8-oxo-dGsn. Simultaneously, pathological analysis of liver biopsy tissues was carried out, and immunohistochemistry was carried out for 8-oxo-Guo, 8-oxo-dGuo and MTH1 protein in some liver injury tissues. We analysed the correlation between the degrees of inflammation and fibrosis and levels of 8-oxo-Gsn and 8-oxo-dGsn. We also analysed the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn with clinical data of HBeAg, HBsAg, and HBV genotype and detected the levels of plasma aspartate aminotransferase, alanine aminotransferase (AST), platelet, alkaline phosphatase, prothrombin time (PT) and HBV DNA, and calculated the aspartate amino transferase-to–platelet ratio index (APRI) score. Nonparametric correlations were used to evaluate the correlation between 8-oxo-Gsn, 8-oxo-dGsn or APRI and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn in patients with liver injury were significantly higher than those of healthy controls (both p?p?=?.013, p?=?.026 and p?=?.049). The receiver operating characteristic curves of 8-oxo-Gsn were 0.696 (0.632–0.759) and 0.731 (0.672–0.790) for inflammatory activity and fibrosis, respectively. Patients with higher levels of urinary 8-oxo-Gsn are more likely to have a high degree of fibrosis and urinary 8-oxo-Gsn may have a great potential in assessing liver fibrosis.     

Oxidative damage of DNA, RNA and their metabolites in leukocytes, plasma and urine of Macaca mulatta: 8-oxoguanosine in urine is a useful marker for aging

The levels of the oxidised forms of guanosine in leukocytes, plasma and urine of Macaca mulatta were determined using a sensitive method based on high-performance liquid chromatography-triple quadruple mass spectrometry (LC-MS/MS). The amounts of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxoGsn), derived from DNA and RNA, respectively, increased with age in leukocytes. The measurement of the free forms of oxidised guanosine revealed similar age-dependent increases of 8-oxo-dGsn and 8-oxoGsn in both plasma and urine, which showed considerably larger amounts of 8-oxoGsn than 8-oxo-dGsn. The 8-oxoGsn content of urine could be a useful biomarker for evaluating aging, as age-dependent increases of 8-oxoGsn are more evident in urine compared to plasma and because urine samples are readily available.     

Oxidative DNA damage by hyperglycemia-related aldehydes and its marked enhancement by hydrogen peroxide

Increased risks of cancers and oxidative DNA damage have been observed in diabetic patients. Many endogenous aldehydes such as 3-deoxyglucosone and glyceraldehyde (GA) increase under hyperglycemic conditions. We showed that these aldehydes induced Cu(II)-mediated DNA damage, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation. GA had the strongest ability to damage DNA, and addition of low concentrations of H2O2 markedly enhanced the DNA damage. GA significantly increased 8-oxodG formation in human cultured cells (HL-60), and H2O2 enhanced it. We conclude that oxidative DNA damage by hyperglycemia-related aldehydes, especially GA, and marked enhancement of DNA damage by H2O2 may participate in diabetes-associated carcinogenesis.     

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.     

Substantial decrease of urinary 8-oxo-7,8-dihydroguanine, a product of the base excision repair pathway, in DNA glycosylase defective mice

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.     

Evidence for attenuated cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine removal in cancer patients

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients.     

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.     

The sacrificial role of easily oxidizable sites in the protection of DNA from damage

It has been suggested that DNA contains sacrificial nucleobase sequences that protect sensitive regions of the genome from oxidative damage. Oxidation of DNA by loss of an electron generates a radical cation that can migrate long distances by hopping. The radical cation can be trapped irreversibly at certain sites (GG steps) by reaction with H₂O or O₂ leading to the formation of lesions (oxidative damage). A series of DNA oligomers that contain regularly spaced GG steps and an 8-oxo-7,8-dihydroguanine (8-oxoG), which serves as a proxy for possibly sacrificial protective low oxidation potential sites, was prepared and analyzed. We find that in certain special sequences of DNA nucleobases that 8-oxoG protects remote GG steps from oxidative damage but that this is not a general phenomenon extending to normal mixed sequence DNA. This is a consequence of the change in the relative rate of charge hopping compared with trapping of the radical cation. When hopping is relatively slow, 8-oxoG exerts no protective effect. Thus, it seems unlikely that low oxidation potential sequences play a meaningful part in protecting mixed sequence DNA from damage.     

Cellular effects of 5-formyluracil in DNA.

5-Formyluracil is a major oxidation product of thymine, formed in DNA in yields comparable to that of 8-oxo-7,8-dihydroguanine by exposure to gamma-irradiation. Whereas the repair pathways for removal and the biological effects of persisting 8-oxo-7,8-dihydroguanine are much elucidated, much less attention has been paid to the cellular implications of 5-formyluracil in DNA. Here we review the present state of knowledge in this important area within research on oxidative DNA damage.     

Formation of 8-nitroguanosine in cellular RNA as a biomarker of exposure to reactive nitrogen species

Reactive nitrogen species, such as peroxynitrite, nitrogen oxides and nitryl chloride, have been implicated as a cause of diverse pathophysiological conditions, including inflammation, neurodegenerative and cardiovascular diseases and cancer. We previously reported that 8-nitroguanine is formed by reactions of guanine or calf-thymus DNA with peroxynitrite in vitro. In the present study, we have studied the formation of 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine in reactions of calf-liver RNA with various reactive nitrogen species. 8-Nitroguanosine in RNA was found to be much more stable than 8-nitro-2' -deoxyguanosine in DNA, which rapidly depurinates to release 8-nitroguanine. Both 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine were formed in calf-liver RNA following exposure to various reactive nitrogen species, such as synthetic peroxynitrite. They were also formed in RNA by reactive species formed from nitric oxide and superoxide anion generated concomitantly from 3-morpholino-sydnonimine (SIN-1) and those formed with myeloperoxidase or horseradish peroxidase in the presence of nitrite and hydrogen peroxide. 8-Nitroguanosine was detected by HPLC with an electrochemical detector in enzymatic hydrolyzates of RNA isolated from human lung carcinoma cells incubated with synthetic peroxynitrite. Our results indicate that 8-nitroguanosine in cellular RNA could be measured as a marker of damage caused by endogenous reactive nitrogen species in tissues and cells.     

8-Nitroguanine formation in oral leukoplakia, a premalignant lesion.

Oral leukoplakia is a premalignant lesion associated with development of oral cancer. To clarify the mechanism of development of oral carcinogenesis from leukoplakia, we examined DNA damage in oral epithelium of biopsy specimens of patients with leukoplakia by immunohistochemical methods. Histological changes, such as epithelial dysplasia and infiltration of inflammatory cells were observed in oral tissues of leukoplakia patients. A double immunofluorescence labeling study demonstrated that the accumulation of mutagenic 8-nitroguanine, an indicator of nitrative DNA damage, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, was apparently observed in the oral epithelium of patients with leukoplakia, whereas little or no immunoreactivity was observed in normal oral mucosa. Expression of inducible nitric oxide synthase (iNOS) was also observed in oral epithelium of leukoplakia patients. Immunoreactivity of 3-nitrotyrosine, an indicator of nitrative stress, was observed in oral epithelial cells and colocalized with 8-nitroguanine. Moreover, proliferating cell nuclear antigen and p53 were expressed in 8-nitroguanine-positive epithelial cells in the basal layer. These results suggest that iNOS-mediated nitrative stress contributes to development of oral carcinogenesis from leukoplakia through DNA damage as well as oxidative stress.     

Association between urinary excretion of cortisol and markers of oxidatively damaged DNA and RNA in humans

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the relationship between 24 h excretion of urinary cortisol and markers of oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, in a sample of 220 elderly men and women (age 65 – 83 years). We found a robust association between the excretion of cortisol and the oxidation markers (R² = 0.15, P<0.001 for both markers). Individuals in the highest quartile of cortisol excretion had a 57% and 61% higher median excretion of the DNA and RNA oxidation marker, respectively, than individuals in the lowest quartile. The finding adds support to the hypothesis that cortisol-induced damage to DNA/RNA is an explanatory factor in the complex relation between stress, aging and disease.     

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.     

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids

A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.     

Exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine: Biomedical properties,mechanisms of action,and therapeutic potential

Abstract—8-Oxo-7,8-dihydroguanine (8-oxo-G) is a key biomarker of oxidative damage to DNA in cells, and its genotox- icity is well-studied. In recent years, it has been confirmed experimentally that free 8-oxo-G and molecules containing it are not merely inert products of DNA repair or degradation, but they are actively involved in intracellular signaling. In this review, data are systematized indicating that free 8-oxo-G and oxidized (containing 8-oxo-G) extracellular DNA function in the body as mediators of stress signaling and initiate inflammatory and immune responses to maintain homeostasis under the action of external pathogens, whereas exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) exhibits pro- nounced antiinflammatory and antioxidant properties. This review describes known action mechanisms of oxidized guanine and 8-oxo-G-containing molecules. Prospects for their use as a therapeutic target are considered, as well as a pharmaceu- tical agent for treatment of a wide range of diseases whose pathogenesis is significantly contributed to by inflammation and oxidative stress.