Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2 '-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2'-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30 mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3 nM (signal-to-noise ratio S/N = 3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4 +/- 18.9 nM versus 14.4 +/- 7.6 nM, P = 0.0004; 23.5 +/- 21.3 mug g(-1) creatinine versus 12.6 +/- 13.2 mug g(-1) creatinine, P = 0.028). (C) 2004 Elsevier B.V. All rights reserved.

Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72 +/- 6.89 mumol/mol creatinine versus 2.33 +/- 2.83 mumol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with nonnoalbuminuria (19.2 +/- 16.8 mug/24 h versus 8.1 +/- 1.7 mug/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA(1)c) (r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride (r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients. (C) 2004 Elsevier B.V. All rights reserved.

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha -tocopherol, or the compounds 17 beta -oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage. (C) 2001 Elsevier Science B.V. All rights reserved.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Effect of some antioxidants on oxidative DNA damage induced by autoxidation of microquantities of sulfite in the presence of Ni(II)/Gly-Gly-L-His

Ni(II)GGH (GGH, glycylglycyl-L-histidine) reacts rapidly with S(IV), in air-saturated solution, to produce Ni(III)GGH. A mechanism is proposed where Ni(III) oxidizes SO(3)(2-) to SO(3)(center dot-), which reacts with dissolved oxygen to produce SO(5)(center dot-), initiating radical chain reactions. DNA strand breaks and 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodGuo) formation were observed in air-saturated solutions containing micromolar concentrations of nickel(II) and S(IV). The efficacies of melatonin, (-)-epigallocatechin-gallate (from green tea), resveratrol, tannic, and ascorbic acids in terms of their inhibitory activities of DNA strand breaks and 8-oxodGuo formation were evaluated.

Lower levels of oxidative DNA damage and apoptosis in lymphocytes from patients undergoing surgery with propofol anesthesia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship among oxidative DNA damage, gastric mucosal density and the relevance of cagA, vacA and iceA genotypes of Helicobacter pylori

The aim of this study was to evaluate the relationship among oxidative DNA damage, density of Helicobacter pylori and the relevance of cagA, vacA and iceA genotypes of H. pylori. Gastric epithelial cells were isolated from 24 uninfected patients, 42 H. pylori infected patients with gastritis, and 61 patients with gastric cancer. Oxidative DNA damage was analyzed by the Comet assay, the density of H. pylori was measured by real-time polymerase chain reaction (PCR), and allelic variants of cagA, vacA and iceA were identified using the PCR. Infected patients by Helicobacter pylori cagA(+), vacAs1 m1 and iceA1 genotype showed higher levels of oxidative DNA damage than infected patients with H. pylori cagA(-), vacAs2 m2 and iceA2 genotypes and uninfected patients. Density of H. pylori did not influence oxidative DNA damage. Our results indicate that H. pylori genotype is more relevant than density for oxidative DNA damage.

Tomato-oleoresin supplement prevents doxorubicin-induced cardiac myocyte oxidative DNA damage in rats

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that reactive oxygen species are involved in DOX-induced cardiotoxicity, antioxidant agents have been used to attenuate its side effects. To determine tomato-oleoresin protection against cardiac oxidative DNA damage induced by DOX, we distributed Wistar male rats in control (C), lycopene (L), DOX (D) and DOX+lycopene (DL) groups. They received corn oil (C, D) or tomato-oleoresin (5 mg/kg body wt. day) (L, DL) by gavage for a 7-week period. They also received saline (C, L) or DOX (4 ma/kg body wt.) (D, DL) intraperitoneally at the 3rd, 4th, 5th, and at 6th week. Lycopene absorption was checked by HPLC. Cardiac oxidative DNA damage was evaluated by the alkaline Comet assay using formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (endo 111). Cardiomyocyte levels of SBs, SBs FPG and SBs Endo III were higher in rats from D when compared to other groups. DNA damage levels in cardiomyocytes from DL were not different when compared to C and L groups. The viability of cardiomyocytes from D or DL was lower than C or L groups (p < 0.01). Lycopene levels (mean +/- S.D. nmol/kg) in saponified hearts were similar between L (47.43 +/- 11.78) and DL (49.85 +/- 16.24) groups. Our results showed: (1) lycopene absorption was confirmed by its cardiac levels; (2) DOX-induced oxidative DNA damage in cardiomyocyte; (3) tomato-oleoresin supplementation protected against cardiomyocyte oxidative DNA damage. (c) 2007 Elsevier B.V. All rights reserved.

Influence of diet on oxidative DNA damage, uracil misincorporation and DNA repair capability

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)