3-Aminobenzamide inhibits cytotoxicity and adhesion of phorbol-ester-stimulated granulocytes to fibroblast monolayer cultures

Damage of 3T3 fibroblasts as induced by short-term co-cultivation with O2(-)-producing granulocytes, stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was compared with that induced by treatment with enzymically generated O2- and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA-stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2- was monophasic. (b) Poly(ADP-ribosyl)ation of nuclear proteins after treatment with TPA-stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3-Aminobenzamide, the most widely used inhibitor of ADP-ribosylation, partially protected target cells from the cytotoxic effects of TPA-stimulated granulocytes, while it enhanced alkylation-induced and O2(-)-induced cytotoxicity. Protection by 3-aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP-ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA-stimulated granulocytes. The unique effect of 3-aminobenzamide in this system appeared to relate to TPA-induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3-aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3-aminobenzamide at the doses applied does not inhibit TPA-induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a 'dilution' of granulocyte-derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.     

Formation of 8-hydroxydeoxyguanosine, hydroxyl free radical adduct of DNA in granulocytes exposed to the tumor promoter, tetradecanoylphorbolacetate

The exposure of human granulocytes to the tumor promoter, tetradecanoylphorbolacetate (TPA), resulted in the accumulation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA of the treated cells. Hydroxyl free radicals react with DNA causing the hydroxylation of guanine at the C-8 position. The modified nucleoside (8-OHdG) cleaved from DNA, was quantitated at subpicomole levels utilizing high pressure liquid chromatography with electrochemical detection (LCED). Superoxide dismutase and catalase caused a marked decrease in the levels of 8-OHdG in the cellular DNA. The level of 8-OHdG formed by TPA stimulation of granulocytes was equivalent to one modified guanine for about every 600 possible guanines in the cellular DNA.     

Effect of lipophilic chelators on oxyradical-induced DNA strand breaks in human granulocytes: paradoxical effect of 1,10-phenanthroline.

Strand breaks can be produced in the DNA of intact granulocytes by a flux of oxyradicals (O2- and H2O2) generated by tetradecanoylphorbol acetate (TPA) or by a flux of H2O2 generated by glucose oxidase. The mechanism by which such breaks are induced is still uncertain. Lipophilic chelators such as dipyridyl and 1,10-phenanthroline (OP) strongly inhibit strand breaks induced by H2O2, presumably because of their ability to chelate intracellular iron. We now report that dipyridyl also partially inhibits strand breaks in TPA-stimulated granulocytes while a "copper-specific" lipophilic chelator, neocuproine, has no effect. As opposed to these effects, OP increases the number of strand breaks in TPA-stimulated granulocytes. Superoxide dismutase (SOD) (but not catalase) partially blocks this increase. Both the cell-impermeable chelator, EDTA, and neocuproine strongly block the increase also. In fact, in the presence of EDTA, OP behaves like dipyridyl and inhibits strand breaks. Preformed OP2-copper(II) complex causes DNA breaks in TPA-stimulated granulocytes. The paradoxical effect of OP may be explained by assuming that OP may form two different metal complexes, a DNA-damaging complex with copper or an inhibitory complex with iron. If copper(II) and O2- are present, the first complex may form and the net effect may be an increase in strand breaks. If the formation of this complex is prevented by SOD, EDTA, or neocuproine, then OP may complex iron and the net effect may be (like dipyridyl) an inhibition of strand breaks. The source of the copper responsible for the formation of OP2-copper complex is unknown.     

8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption

8-Hydroxydeoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal and human studies. However, controversial data exist on the relationship between 8-OHdG formation and age, sex and tobacco smoking in humans, while few or no data are available on other exposures such as environmental tobacco smoke, alcohol, coffee and tea consumption. We investigated the level of 8-OHdG in DNA from peripheral leukocytes among 102 healthy adults living in Brescia province, North Italy, aged 25-45 (mean: 35.2 years), of which 51 were males. 8-OHdG levels expressed as a ratio to total deoxyguanosine (8-OHdG/106 dG) in DNA showed wide interindividual variation, the highest value (63.8) being 6. 2-fold greater than the lowest (10.3). Current smokers showed lower mean 8-OHdG values than subjects who never smoked (29.3 and 34.0, respectively, p<0.05), and an inverse relationship was found between 8-OHdG and lifetime smoking, which was independent of age, sex and body mass index. An inverse relationship was also found with coffee drinking while no association was observed with alcohol and tea consumption, exposure to environmental tobacco smoke and use of vitamins in all subjects, and with use of oral contraceptives in females. The inverse relationship between smoking status and 8-OHdG levels could be explained by the presence of efficient repair processes for the oxidative damage induced by smoking. In this study, the smokers were relatively young (77% were less than 40 years) and only 7% smoked 30 or more cigarettes a day. In conclusion, it would appear that 8-OHdG levels in leukocytes may not provide a sensitive marker of exposure to tobacco smoking.     

Muscle soreness-induced reduction in force generation is accompanied by increased nitric oxide content and DNA damage in human skeletal muscle

We examined the effect of exercise-induced muscle soreness on maximal force generation, tissue nitric oxide (NO) and 8-hydroxydeoxyguanosine (8-OHdG) content in human skeletal muscle. Female volunteers were assigned to control (C) and muscle soreness (MS) groups (n = 6 in each). MS group performed 200 eccentric muscle actions of the rectus femoris to induce muscle soreness. Maximal force generation was measured 24 h before and after exercise in both groups. Needle biopsy samples were assayed for NO content with electron spin resonance spectroscopy after ex vivo spin trapping, and 8-OHdG content were measured with an enzyme-linked immuno assay. Maximal force decreased by 11+/-5.4% (p < .05) 24 h after exercise in MS group. Muscle soreness increased NO and 8-OHdG contents from their control values of 0.39+/-0.08 arbitrary units and 0.035+/-0.004 pmol/micromol DNA to 0.96+/-0.05 (p < .05) arbitrary units and 0.044+/-0.005 (p < .05) pmol/micromol DNA, respectively. This is the first demonstration that muscle soreness-induced decrease in maximal force generation is a result of an increase in muscular NO content and associated with enhanced formation of 8-OHdG in human skeletal muscle.     

Increased DNA Oxidation and Decreased Levels of Repair Products in Alzheimer's Disease Ventricular CSF

Abstract : One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solidphase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations ( p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples ( p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.     

Oxidative stress and altered antioxidant defenses in children with acute exacerbation of atopic dermatitis

The underlying mechanisms of skin inflammation in atopic dermatitis (AD) are not completely understood. The purpose of the present study was to examine the involvement of oxidative stress and antioxidant defenses in children with acute exacerbation of AD. We studied 13 children who were hospitalized for acute exacerbation of AD with purulent skin infection by Staphylococcal aureus (age, 1.5 to 10.0 years), and 28 age-matched healthy subjects (controls). Urine samples obtained from the patients on admission, on 2nd and 7th-9th hospital days, as well as from the controls were analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (a marker of oxidative DNA damage), acrolein-lysine adducts (a marker of lipid peroxidation), bilirubin oxidative metabolites (BOM) (a marker of antioxidant activity of bilirubin under oxidative stress) and nitrite/nitrate (NO(x)(-)) (a marker of endogenous nitric oxide production). Of these, urinary concentrations of 8-OHdG, acrolein-lysine adducts and BOM, but not NO(x)(-), were significantly higher in AD children on admission than those in control subjects. Response to treatment was associated with significant falls in the concentrations of 8-OHdG and acrolein-lysine adducts. Urinary concentrations of acrolein-lysine adducts, but not 8-OHdG, were still significantly higher in AD patients on the 7th-9th hospital day relative to the control. Urinary BOM remained almost constant and significantly high in AD children during hospitalization. Our findings indicate that oxidative stress and altered antioxidant defenses are involved in the pathophysiology of acute exacerbation of AD, and that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

1 alpha, 25-dihydroxyvitamin D3 modulates the growth of 3T3 cells and human skin fibroblasts stimulated by platelet-derived growth factor

We investigated the effect of 1 alpha,25-dihydroxyvitamin D3 (1,25 (OH)2 vit D3) on the 3H-thymidine uptake by Balb/c 3T3 cells and by human skin fibroblasts stimulated by normal human serum or by purified PDGF. We found an inhibitory effect of 1,25 (OH)2 vit D3 on the DNA synthesis of Balb/c 3T3 cells grown in the presence of human serum as well as in the presence of PDGF. At 5% human serum this effect is minimal at 10(-12) M 1,25 (OH)2 vit D3 and is maximal at 10(-9) M. On the DNA synthesis of human fibroblasts stimulated by human serum or by PDGF a modulatory effect of 1,25 (OH)2 vit D3 was shown. On these cells the vitamin had a stimulatory effect between 10(-11) and 10(-9) M and an inhibitory effect at very high concentrations (10(-7) M). Our results suggested that the effect of 1,25 (OH)2 vit D3 on fibroblast DNA synthesis could be mediated by interactions with its specific intracellular receptor. 1,25 (OH)2 vit D3 had no any action on the growth of human fibroblasts stimulated by fibroblast growth factor.     

Levels of 8-hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany

Asbestos fibers have genotoxic effects and are a potential carcinogenic hazard to occupationally exposed workers. The ability of inhaled asbestos fibers to induce the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the DNA of white blood cells (WBC) of workers highly exposed at the workplace has been studied. The 8-OHdG adduct level of asbestos-exposed workers was significantly increased (p<0.001) compared to that in the control group in all three years of the study. Asbestos-exposed individuals showed a mean value of 2.61+/-0.91 8-OHdG/10(5) dG (median 2.49, n=496) in 1994-1995, 2.96+/-1.10 8-OHdG/10(5) dG (median 2.76, n=437) in 1995-1996 and 2.55+/-0.56 8-OHdG/10(5) dG (median 2.53, n=447) in 1996-1997. For the control subjects, a mean of 1.52+/-0.39 (median 1.51, n=214) was determined. The results indicate that human DNA samples from exposed individuals contain between 1.7 times and twice the level of oxidative damage relative to that found in control samples in all 3 years of the study. The studies presented here show that asbestos exposure can result in oxidative DNA damage. Our data confirm that oxidative DNA damage occurs in the WBC of workers highly exposed to asbestos fibers, thus supporting the hypothesis that asbestos fibers damage cells through an oxidative mechanism. These in vivo findings underline the importance of oxidative damage in asbestos-induced carcinogenesis and highlight the need for exploring the molecular basis of asbestos-induced diseases, and for more effective diagnosis, prevention and therapy of mesothelioma, lung cancer and pulmonary fibrosis. In addition, preventive and therapeutic approaches using antioxidants may be relevant.     

Diet modification affects DNA oxidative damage in healthy humans

DNA 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a promising biomarker for oxidative damage. We assessed its responsiveness to diet in 32 nonsmoking, healthy subjects (12 male, 20 female) aged 31+/-7.6 years. They consumed two liquid formula diets (Ensures) as the sole source of nutrition for 10-d in a randomized crossover design, with 5-d control solid food diets as washout before each liquid diet period. Reformulated Ensure (Re-En) had a vitamin E/ PUFA of 3.5 compared to standard Ensure (En) of 1.1. We hypothesized that subjects would have lower leukocyte 8-OHdG/deoxyguanosine (dG) ratios while consuming Re-En compared to En. But 8-OHdG/dG ratios did not change with the consumption of either Re-En or En. The mean ratios of 8-OHdG/dG after 10 days of Re-En and En consumption were (2.12+/-0.68)x10(-5) and (2.16+/-0.63)x10(-5), respectively. However, there was a 22% decrease in 8-OHdG/dG by the end of the study and a significant downward trend of leukocyte 8-OHdG among all subjects throughout all nutrient-rich diet phases as the study progressed (Test for trend: p = .04; paired t-test: p = .07). Because all the experimental diets provided antioxidant nutrients at higher quantities than typically consumed by a U.S. age-matched population, this study adds to the few in vivo studies that show a decrease in DNA damage in healthy nonsmoking subjects through dietary intervention.     

Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter

Exposure to ambient particulate matter has been reported to be associated with increased rates of lung cancer. Previously we showed that total suspended particulate matter (PM) induces oxidative DNA damage in epithelial lung cells. The aim of the present study was to further investigate the mechanism of PM-induced DNA damage, in which soluble iron-mediated hydroxyl radical (.OH) formation is thought to play a crucial role. Using electron spin resonance (ESR) we showed that PM suspensions as well as their particle-free, water-soluble fractions can generate .OH in the presence of hydrogen peroxide (H2O2), an effect which was abrogated by both deferoxamine and catalase. In addition, PM was also found to induce the .OH-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the presence of H2O2 as assessed by dot-blot analysis of calf thymus DNA using an 8-OHdG antibody. In human alveolar epithelial cells (A549), both PM suspensions and the particle-free soluble fraction elicited formation of DNA strand breaks (comet-assay). Unlike the acellular DNA assay, in epithelial cells the DNA-damaging capacity of the particle suspensions appeared to be stronger than that of their corresponding particle-free filtrates. In conclusion, our findings demonstrate that the water-soluble fraction of PM elicits DNA damage via transition metal-dependent .OH formation, implicating an important role of H2O2. Moreover, our data indicate that direct 'particle' effects contribute to the genotoxic hazard of ambient particulate matter in lung target cells.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Association of hepatitis virus infection,alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

Evidence of oligonucleotides containing 8-hydroxy-2'-deoxyguanosine in human urine

The product of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of <30-55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods.

8-Hydroxy-2'-deoxyguanosine (8OH2'dG) is a principal stable marker of hydroxyl radical damage to DNA. It has been related to a wide variety of disorders and environmental insults, and has been proposed as a useful systematic marker of oxidative stress. Analytic procedures for 8OH2'dG in DNA digests are well established; however, routine measurement of free 8OH2'dG in other body fluids such as urine or plasma has been problematic. This has hindered its evaluation as a general clinical, therapeutic monitoring, or environmental assessment tool. Therefore, we developed a liquid chromatography electrochemical column-switching system based on the use of the unique purine selectivity of porous carbon columns that allows routine accurate measurement of 8OH2'dG in a variety of biologic matrices. This paper describes the rationale of the system design and the protocols developed for 8OH2'dG in urine, plasma, cerebrospinal fluid, tissue, DNA, saliva, sweat, kidney dialysis fluid, foods, feces, culture matrix, and microdialysates. Concentrations in both human and animal body fluids and tissues are reported. The system performance is discussed in the context of a 1-year evaluation of the methods applied to approximately 3600 samples, using internal quality control and external blind testing to determine long-term accuracy. The methods are reliable and accurate, and therefore should prove useful in assessing the role and utility of oxidative DNA damage in aging and human illness.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

Chloroacetonitrile (CAN) induces glutathione depletion and 8-hydroxylation of guanine bases in rat gastric mucosa

Chloroacetonitrile (CAN) is detected in drinking-water supplies as a by-product of the chlorination process. Gastroesophageal tissues are potential target sites of acute and chronic toxicity by haloacetonitriles (HAN). To examine the mechanism of CAN toxicity, we studied its effect on glutathione (GSH) homeostasis and its impact on oxidative DNA damage in gastric mucosal cells of rats. Following a single oral dose (38 or 76 mg/Kg) of CAN, animals were sacrificed at various times (0-24 h), and mucosa from pyloric stomach were collected. The effects of CAN treatment on gastric GSH contents and the integrity of genomic gastric DNA were assessed. Oxidative damage to gastric DNA was evaluated by measuring the levels of 8-Hydroxydeoxyguanosine (8-OHdG) in hydrolyzed DNA by HPLC-EC. The results indicate that CAN induced a significant, dose- and time-dependent, decrease in GSH levels in pyloric stomach mucosa at 2 and 4 hours after treatment (56 and 39% of control, respectively). DNA damage was observed electrophoretically at 6 and 12 hours following CAN administration. CAN (38 mg/Kg) induced significant elevation in levels of 8-OHdG in gastric DNA. Maximum levels of 8-OHdG in gastric DNA were observed at 6 hours after CAN treatment [9.59+/-0.60 (8-OHdG/10(5)dG) 146% of control]. When a high dose of CAN (76 mg/Kg) was used, a peak level of 8-OHdG [11.59+/-1.30 (8-OHdG/10(5)dG) 177% of control] was observed at earlier times (2 h) following treatment. When CAN was incubated with gastric mucosal cells, a concentration-dependent cyanide liberation and significant decrease in cellular ATP levels were detected. These data indicate that a mechanism for CAN-induced toxicity may be partially mediated by depletion of glutathione, release of cyanide, interruption of the energy metabolism, and induction of oxidative stress that leads to oxidative damage to gastric DNA.