Time and concentration dependence of Fenton-induced oxidation of dG

The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2'-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8,5'-cyclo-2'-deoxyguanosine (8,5'cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5' cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodG a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5' cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG alone.     

Urinary 8-hydroxy-2′-deoxyguanosine (8-oxodG) level can predict acute renal damage in young children with urinary tract infection

Abstract

Background: There are no good biomarkers to predict renal parenchymal involvement in children with urinary tract infection (UTI).

Methods: Children (N?=?73) younger than 5 years with UTI were enrolled. Urinary levels of 8-hydroxy-2′-deoxyguanosine (8-oxodG) and total antioxidant capacity (TAC) were checked as markers of oxidative stress and antioxidant capacity, respectively. Tc99m-dimercaptosuccinic acid (DMSA) renal scintigraphy was used to find evidence of renal involvement.

Results: Patients with positive DMSA findings had higher levels of urinary 8-oxodG (p?=?0.003) and higher urinary TAC (p?=?0.001) than patients with normal DMSA findings.

Conclusions: High level of urinary 8-oxodG may be a risk factor of severe renal damage.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

Controlled oxidation of calf thymus DNA to produce standard samples for 8-Oxodeoxyguanosine analysis; Effects of freeze-drying,storage and hydrolysis conditions

Calf thymus DNA containing defined levels of 8-hydroxy-2′-deoxyguanosine (8-oxodG) was prepared by treatment with visible light in the presence of photosensitiser Ro 19-8022. The DNA was checked for stability; after freeze-drying, the amount of 8-oxodG did not increase during 6 weeks' storage at room temperature. However, freeze-drying itself can introduce additional oxidative damage. Two enzymic hydrolysis regimes (DNase I, phosphodiesterases I and II, and alkaline phosphatase; or P1 nuclease and alkaline phosphatase) give similar values for 8-oxodG.     

Comparison of Results from Different Laboratories in Measuring 8-oxo-2′-deoxyguanosine in Synthetic Oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 &#119 M, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage

We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2′-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within ±40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, ³²P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended.     

Methodological considerations and factors affecting 8-hydroxy-2′-deoxyguanosine analysis

Oxidative stress is related to a number of diseases due to the formation of reactive oxygen species (ROS). There are also several substances found in the occupational environment or as life style related situations that generates ROS. A stable biomarker for oxidative stress on DNA is 8-hydroxy-2′-deoxyguanosine (8-OH-dG).

A potential problem in the work-up and analysis of 8-OH-dG is oxidation of dG with false high levels as a result of analysis. This paper summarizes and discusses some of the critical moments in terms of auto-oxidation. The removal of transition metals, low temperatures, absence of isotopes (or 2′-deoxyguanosine) and incubation times are all important factors. Removal of oxygen is complicated while the problem is reduced if a nitroxide (TEMPO) is added during work-up. Certain reducing agents and enzymes could be critical if added during work-up.

The application of the ³²P-HPLC method to analyze 8-OH-dG is discussed. The ³²P-HPLC method is suitable for 8-OH-dG analysis and avoids several factors that oxidizes dG by removal of dG before addition of isotopes. Factors of crucial importance (columns, eluents, gradients and detection of ³²P) for the analysis of 8-OH-dG are commented upon and certain recommendations are made to make it possible to apply the ³²P-HPLC methodology for this type of analysis.     

Suppression of 8-Oxo-2′-deoxyguanosine Formation and Carcinogenesis Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters by Esculetin and Esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2′-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30?min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4?h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy,full-term newborns

The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2′-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7–8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.     

Synthesis and Evaluation of Pyrrole Polyamide- 2′-Deoxyguanosine 5′-Phosphate Hybrid

Pyrrole polyamide-2′-deoxyguanosine 5′-phosphate hybrid (Hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was found to exhibit dose-dependent inhibition of cell growth.     

Elevated urinary levels of 8-oxo-2′-deoxyguanosine, (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines,and 8-iso-prostaglandin F2α as potential biomarkers of oxidative stress in patients with prediabetes

Prediabetes is the preclinical stage of type 2 diabetes mellitus (T2DM) with intermediate state of hyperglycemia. Hyperglycemia results in a state of oxidative stress, which may contribute to the production of insulin resistance, β-cell dysfunction and long-term complications of diabetes. Novel approaches are required for prevention and treatment of diabetes. New biomarkers that can be used in risk stratification and therapy control as supplementary to current parameters are needed. These biomarkers may facilitate a more individualized and sufficient treatment of diabetes. Therefore, the aim of this study was to investigate the levels of oxidatively induced DNA damage products, 8-oxo-2′-deoxyguanosine (8-oxo-dG) (also known as 8-OH-dG), (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines (R-cdA and S-cdA), and the lipid peroxidation product 8-iso-prostaglandin F_2α (8-iso-PGF_2α) as reliable oxidative stress markers in patients with prediabetes or T2DM in comparison with healthy volunteers. Urine samples were collected from these subjects. Absolute quantification of 8-oxo-dG, R-cdA, S-cdA and 8-iso-PGF_2α was achieved by liquid chromatography-isotope dilution tandem mass spectrometry. The levels of 8-oxo-dG, S-cdA and 8-iso-PGF_2α were significantly greater in prediabetes patients than those in healthy volunteers. T2DM patients also had higher levels of 8-oxo-dG than healthy volunteers. No statistically significant difference was observed for R-cdA levels. 8-Oxo-dG levels positively correlated with R-cdA and S-cdA levels for prediabetes and newly diagnosed T2DM. S-cdA levels and HbA1c were found negatively correlated in prediabetes patients. Also 8-iso-PGF_2α levels and HbA1c were found negatively correlated in prediabetes patients. These results indicate that oxidatively induced macromolecular damage appears before the establishment of T2DM. Thus, our data suggest that oxidatively induced DNA damage and lipid peroxidation products that were found to be elevated in prediabetic stage may be used as early disease markers in patients at risk for T2DM.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

DNA Repair: Insights from Urinary Lesion Analysis

Due to various confounding factors, namely dietary contribution and cell death, measurement of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) has long been considered to be no more than a marker of generalised oxidative stress. Indeed, the action of no single enzyme has been reported to excise 8-oxodG from DNA. However, analysis of recent research has suggested that these confounders may be circumvented, which, combined from work from the authors' laboratory, indicates that urinary 8-oxodG has the potential to become a most important marker of oxidative damage to, and repair of, DNA.     

Detection of extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine as a biomarker of oxidative damage in X-irradiated fibroblast cultures: optimization of analytical procedure

We have developed a simple methodology, based on single-step solid-phase extraction followed by isocratic high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD), to determine extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in culture supernatants of normal human dermal fibroblasts. A standard addition method, using externally added 8-oxodG (0.5 and 1 pmol) was employed to eliminate matrix effects arising from the chemically complex, protein-rich medium. Secondly, applying this procedure to X-ray irradiated fibroblasts, we report a significant twofold increase in the levels of 8-oxodG at the radiobiologically relevant dose of 6 Gy. This suggests that extracellular 8-oxodG might be a useful biomarker for oxidative stress following moderate doses of X-irradiation.     

Iron-induced oxidative DNA damage in rat sperm cells in vivo and in vitro

We investigated whether acute iron intoxication causes oxidative DNA damage, measured in terms of 7-hydro-8-oxo-2′-deoxyguanosine, 8-oxodG, in nuclear DNA in testes and epididymal sperm cells in vivo and in vitro in rats. In addition, we investigated levels of the modified nucleoside in liver and kidney and measured its urinary excretion.

Sperm cells were isolated from the epididymides and the testes cells were isolated after homogenisation. In vitro, the sperm and testes cells were incubated with increasing concentrations of FeCl₂ ranging from 0 to 600 μM. The median (range) levels of 8-oxodG/10⁵ dG in the epididymal sperm cells increased from 0.48 (0.42–0.90) to 15.1 (11.4–17.6) (p < 0.05), whereas the level rose from 0.63 (0.22–0.81) to 8.8 (4.5–11.6) (p < 0.05) at 0 and 600 μM, respectively, in the testicular cells.

In vivo groups of 7–8 rats received 0, 200 or 400 mg iron/kg as dextran i.p. After 24h, epididymal sperm cells, testes, kidneys and liver were collected for analysis. Kidney and sperm DNA showed a significant increase in 8-oxodG in the iron-treated animals. The median (range) values of the 8-oxodG/10⁵ dG in the epididymal sperm cells rose from 0.66 (0.38–1.09) to 1.12 (0.84–5.88) (p < 0.05) at 0 and 400 mg iron/kg, respectively, whereas the values in the testes and liver showed no significant change. In the kidneys the 8-oxodG/10⁵ dG median (range) values were 0.98 (0.73–1.24), 1.21 (1.13–1.69) and 1.34 (1.12–1.66) after 0, 200 and 400 mg iron/kg, respectively (p < 0.05).

The 8-oxodG-excretion rate was measured in 24 h urine before and after iron treatment. The rate of urinary 8-oxodG excretion increased from 129 (104–179) pmol/24 h before treatment to 147 (110–239) pmol/24h after treatment in the group receiving 400 mg iron/kg (p < 0.05).

The results indicate that acute iron intoxication may increase oxidative damage to sperm and kidney DNA.     

Nonylphenol exposure is associated with oxidative and nitrative stress in pregnant women

ABSTRACT

Animal studies have shown that exposure to nonylphenol (NP) increases oxidative/nitrative stress, but whether it does so in humans is unknown. This study examines prenatal exposure to NP and its effects on oxidatively/nitratively damaged DNA, lipid peroxidation, and the activities of antioxidants. A total of 146 urine and blood specimens were collected during gestational weeks 27–38 and hospital admission for delivery, respectively. Urinary NP was analyzed by high-performance liquid chromatography (HPLC). Urinary biomarkers of oxidatively/nitratively damaged DNA and lipid peroxidation, including 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), 8-nitroguanine (8-NO₂Gua), 8-iso-prostaglandin F_2α (8-isoPF_2α) and 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA), were simultaneously analyzed using isotope-dilution liquid-chromatography/electron spray ionization tandem mass spectrometry. The activities of maternal plasma superoxide dismutase and glutathione peroxidase were analyzed by enzyme-linked immunosorbent assay. Urinary NP level was significantly associated with 8-oxodG and 8-NO₂Gua levels in late pregnancy, suggesting that NP may enhance oxidatively and nitratively damaged DNA. The adjusted odds ratios for high 8-oxodG level exhibited a significantly dose–response relationship with NP levels, stratified into four quartiles. 8-oxodG appears to be a more sensitive and effective biomarker of NP exposure than 8-NO₂Gua. These relationships suggest NP may play a role in the pregnancy complications.     

Exercise-induced oxidatively damaged DNA in humans: evaluation in plasma or urine?

Physical exercise can induce oxidative damage in humans. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely known biomarker of DNA oxidation, which can be determined in blood and urine. The aim of the present study was to compare these two biological fluids in terms of which is more suitable for the estimation of the oxidative damage of DNA by measuring the concentration of 8-OHdG one hour after maximal exercise by enzyme immunoassay. The concentration of 8-OHdG increased with exercise only in plasma (p?<?0.001), and values differed between exercise tests in both plasma and urine (p?<?0.05). In conclusion, plasma appears to be more sensitive to exercise-induced 8-OHdG changes than urine and, hence, a more appropriate medium for assessing oxidative damage of DNA, although the poor repeatability of the measurement needs to be addressed in future studies