Urinary thymine dimers and 8-oxo-2′-deoxyguanosine in psoriasis

Psoralen in conjunction with UVA (PUVA) is perhaps the most effective treatment for psoriasis. It is, however, a risk factor for skin cancer in these patients and there is a need to develop non-invasive assays reflective of treatment-induced DNA damage. We report here the assessment of two important lesions, thymine dimer (T<>T) and 8-oxo-2'-deoxyguanosine (8-OHdG), in the urine of psoriasis patients. It was found that, once corrected for urine concentration, the psoriatic group had significantly higher (P<0. 0001) urinary levels of thymine dimers compared to the control group. No significant differences in urinary 8-OHdG levels were noted between the psoriatic, atopic dermatitis and control groups. Therefore biomonitoring of therapy from the very start with this simple and non-invasive assay could perhaps be an effective measure of the risk involved with the treatment allowing optimization for minimal-risk therapy.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker in type 2 diabetes

The increasing prevalence of diabetes together with the associated morbidity and mortality calls for additional preventive and therapeutic strategies. New biomarkers that can be used in therapy control and risk stratification as alternatives to current methods are needed and can facilitate a more individualized and sufficient treatment of diabetes. Evidence derived from both epidemiological and mechanistic studies suggests that oxidative stress has an important role in mediating the pathologies of diabetic complications. A marker of intracellular oxidative stress that potentially could be used as a valuable biomarker in diabetes is the DNA oxidation marker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), which can be assessed noninvasively in the urine, with minimal discomfort for the patient. In this review the analytical validity of 8-oxodG is addressed by highlighting important methodological issues. The available epidemiological evidence regarding urinary 8-oxodG and type 2 diabetes is presented. A possible role for DNA oxidation in cancer development in type 2 diabetes patients is discussed, followed by an evaluation of the potential of urinary 8-oxodG as a clinical biomarker in type 2 diabetes.     

Simultaneous measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine by HPLC-MS/MS

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54±2.07 vs. 6.70±1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16±0.35 vs. 1.35±0.50 nmol/mmol creatinine, p=0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r_p=0.44), BMI (r_p=-0.27) and nicotine derivatives (r_p=0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r_p=0.36), nicotine derivatives (r_p=0.29), BMI (r_p=-0.24) on the other.     

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

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

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

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

Biological role of 8-oxo-2'-deoxyguanosine

The review presents currently available data on the biological role of 8-oxo-2’-deoxyguanosine. This compound for a long time has been used successfully as a biomarker of oxidative stress and diseases associated with it. However, in recent years, an increasing number of publications has appeared reporting that biological role of 8-oxo-dG is not only a mutagenic one; it also is involved in the regulation of gene expression, in some processes of DNA repair, in the control of inflammatory and autoimmune reactions, and in the activation of antioxidant systems. Probably, 8-oxo-2’-deoxyguanosine could prospectively be used as a drug.     

Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA

Oxidatively damaged DNA may be important in carcinogenesis. 8-Oxo-7,8-dihydroguanine (8-oxoGua) is an abundant and mutagenic lesion excised by oxoguanine DNA glycosylase 1 (OGG1) and measurable in urine or plasma by chromatographic methods with electrochemical or mass spectrometric detectors, reflecting the rate of damage in steady state. A common genetic OGG1 variant may affect the activity and was associated with increased levels of oxidized purines in leukocytes without apparent effect on 8-oxoGua excretion or major change in cancer risk. 8-OxoGua excretion has been associated with exposure to air pollution, toxic metals, tobacco smoke and low plasma antioxidant levels, whereas fruit and vegetable intake or dietary interventions showed no association. In rodent studies some types of feed may be source of 8-oxoGua in collected urine. Of cancer therapies, cisplatin increased 8-oxoGua excretion, whereas radiotherapy only showed such effects in experimental animals. Case-control studies found high excretion of 8-oxoGua in relation to cancer, dementia and celiac disease but not hemochromatosis, although associations could be a consequence rather than reflecting causality of disease. One prospective study found increased risk of developing lung cancer among non-smokers associated with high excretion of 8-oxoGua. Urinary excretion of 8-oxoGua is a promising biomarker of oxidatively damaged DNA.     

Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry

A method for the determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and urine by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry is described. For the urine samples there is no sample preparation except for addition of buffer and internal standards followed by redissolvation of precipitate containing 8-oxo-2'-deoxyguanosine and a centrifugation step before the samples are injected onto the HPLC column. The detection limit for 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine is approximately 0.3 nM corresponding to 7.5 fmol injected. Long runs, that is, > 50 samples, can be analyzed with only minimal loss of sensitivity. The concentrations excreted into urine samples from humans are between 1 and 100 nM for 8-oxo-2'-deoxyguanosine and below 0.3 nM for 8-oxo-2'-deoxyadenosine. In calf thymus DNA levels down to about 1 oxidized guanosine and adenosine per 10(6) unmodified bases can be detected. High levels of 8-oxo-2'-deoxyguanosine were found, 30 per 10(6) 2'-deoxyguanosine, levels of 8-oxo-2'-deoxyadenosine are at or below the detection limit. These findings indicate that High Performance Liquid Chromatography-Tandem Mass Spectrometry is a highly sensitive and specific method for analysis of oxidative DNA modifications in tissue as well as for analysis of excretion of oxidized nucleotides into urine that ensures a minimum artifact formation.     

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CVof less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10(6) Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.     

Anti-8-oxo-2'-deoxyguanosine phage antibodies: isolation, characterization, and relationship to disease states

We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2'-deoxyguanosine (8-oxodG). One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light. In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg). The nucleotide sequence of the 175G V(H) gene segment was 98% homologous to the published V(H) sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE). Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G. The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity. 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA.     

Leukocyte 8-oxo-7,8-dihydro-2'-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane--an iron chelator--was proven to decrease free radical production and anthracycline cardiotoxicity. In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) relative to 2'-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry. Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3 microM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant (rho =-0.517, p = 0.023). This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.     

Harmonising measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and urine

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2'-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2'-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement.     

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine oxidation

8-Oxo-7,8-dihydroguanine (8-oxoGua), an important biomarker of DNA damage in oxidatively generated stress, is highly reactive towards further oxidation. Much work has been carried out to investigate the oxidation products of 8-oxoGua by one-electron oxidants, singlet oxygen, and peroxynitrite. This report details for the first time, the iron- and copper-mediated Fenton oxidation of 8-oxoGua and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Oxidised guanidinohydantoin (Gh(ox)) was detected as the major product of oxidation of 8-oxoGua with iron or copper and hydrogen peroxide, both at pH 7 and pH 11. Oxaluric acid was identified as a final product of 8-oxoGua oxidation. 8-oxodGuo was subjected to oxidation under the same conditions as 8-oxoGua. However, dGh(ox) was not generated. Instead, spiroiminodihydantoin (Sp) was detected as the major product for both iron and copper mediated oxidation at pH 7. It was proposed that the oxidation of 8-oxoGua was initiated by its one-electron oxidation by the metal species, which leads to the reactive intermediate 8-oxoGua (+), which readily undergoes further oxidation. The product of 8-oxoGua and 8-oxodGuo oxidation was determined by the 2'-deoxyribose moiety of the 8-oxodGuo, not whether copper or iron was the metal involved in the oxidation.     

Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs.

Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA. One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA. During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8). The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above). Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells.     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and genetic polymorphisms in breast cancer patients

Reactive oxygen species (ROS) causes damage to DNA, but the role of ROS in breast carcinoma is still not clear. The objective of this study was to measure the urinary 8-OHdG levels of breast cancer patients at each stage of carcinogenesis and assess its association with the development of breast cancer. Sixty patients with malignant breast tumors were matched with 60 control subjects of the same ages in this case control study. Urinary 8-OHdG levels were significantly higher among breast cancer patients than among the control subjects, after making adjustments for confounders such as smoking, coffee consumption and use of oral contraceptives. The breast cancer patients were divided into three groups based on the stages of their cancer; urinary 8-OHdG levels decreased with each stage of breast carcinoma. Using multiple regression and logistic models adjusted for other covariates, urinary 8-OHdG levels significantly correlated with the development of breast cancer. However, it was found that breast cancer was not significantly influenced by CYP1A1, CYP1M1 or NAT2 polymorphisms. In conclusion, it was found that oxygen radical generation occurred within carcinoma cells, but the role of polymorphism of specific genes in the development of breast cancer should be evaluated.     

Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2'-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated on the workday. Repeated measures ANOVA and multifactorial ANCOVA with CYP1A2 activity, age and UDS as covariates were used for statistical evaluation. On the workday, the 8-oxodG excretion was 190+/-108 and 146+/-89 pmol/kg 24 h in the bus drivers from central and the suburban/rural areas Copenhagen, respectively (p<0.05). The 8-oxodG excretion was not significantly different between the workday and the day off. CYP1A2 activity was not affected by driving area but was correlated with the 8-oxodG excretion on the workday (r=0.53; p<0.05). UDS was not significantly affected by driving area or correlated with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggests that exposure to ambient air pollution causes oxidative damage to DNA. This effect may be modified by the activity of CYP1A2 or a coregulated enzyme.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes

The Fusarium mycotoxin, 4-deoxynivalenol, is an abundant, natural contaminant of corn and wheat. 8-Oxo-12,13-epoxytrichothecenes related to 4-deoxynivalenol were synthesized; they either lacked the 7-hydroxyl but contained a hydroxyl at C-4 (7-deoxynivalenol) or lacked substituents at C-3 and C-7 (3,7-dideoxynivalenol). The ability of these synthetic analogs and their acetylated derivatives to inhibit protein synthesis by cultured mammalian cells was compared to that of 4-deoxynivalenol. Whereas the 50% inhibitory dose (ID50) for murine erythroleukemia cells was about 1 microgram/ml for 4-deoxynivalenol and 3,7-dideoxynivalenol, all of the other analogs were at least 10-fold less potent. When tested at their ID50 dose, all of the 8-oxotrichothecenes, except 4-deoxynivalenol and 3,7-dideoxynivalenol, caused polysome 'run-off', indicating that, at this dose, they are inhibitors of polypeptide chain initiation. With 4-deoxynivalenol and 3,7-dideoxynivalenol, polysomes remained at control levels indicating that these toxins prevent polypeptide chain elongation. From these results and comparisons to previous studies of 8-oxo-12,13-epoxytrichothecenes (trichothecolone, trichothecin, nivalenol and fusarenone X), trichothecenes with substituents at both C-3 and C-4 predominantly inhibit polypeptide chain initiation, whereas those lacking one substituent at either site are inhibitors of chain elongation.