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1.
Chemistry-based studies on oxidative DNA damage: formation,repair, and mutagenesis 总被引:10,自引:0,他引:10
Kasai H 《Free radical biology & medicine》2002,33(4):450-456
Since the discovery of 8-OH-dG formation, various aspects of oxidative DNA damage have been studied. For example, 2-OH-dA and a glyoxal-dG adduct were discovered as new types of oxidative DNA damage; 2-OH-dATP was found to induce mutations and to be a good substrate of a nucleotide sanitization enzyme, the MTH1 protein; and efforts were continued to establish standard methodologies for 8-OH-dG analyses in urine and cellular DNA. By these studies, we found solid chemistry-based approaches were often useful to clarify the biological phenomena. 相似文献
2.
Nakashima T Okada T Asahi J Yamashita A Kawai K Kasai H Matsuno K Gamou S Hirano T 《Mutation research》2008,654(2):138-144
Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd. 相似文献
3.
Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine. 总被引:2,自引:0,他引:2
The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2'-deoxyguanosine; 8-OHdG) by quantitative gel electrophoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these two different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by approximately 8-fold and 8-OHdG production by approximately 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron. 相似文献
4.
Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds. 总被引:5,自引:0,他引:5
S Kawanishi S Inoue S Oikawa N Yamashita S Toyokuni M Kawanishi K Nishino 《Free radical biology & medicine》2001,31(1):108-116
DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in cultured cells exposed to nickel compounds were measured by using a pulsed field gel electrophoresis technique. Among nickel compounds (Ni(3)S(2), NiO (black), NiO (green), and NiSO(4)), only Ni(3)S(2), which is highly carcinogenic, induced lesions of both double- and single-stranded DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured HeLa cells with Ni(3)S(2) (10 microg/ml) induced a 1.5-fold increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) compared with control, whereas NiO (black), NiO (green), and NiSO(4) did not enhance the generation of 8-OH-dG. Intratracheal instillation of Ni(3)S(2), NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs significantly. NiSO(4) induced a smaller but significant increase in 8-OH-dG. Histological studies showed that all the nickel compounds used induced inflammation in lungs of the rats. Nitric oxide (NO) generation in phagocytic cells induced by Ni(3)S(2), NiO(black), and NiO(green) was examined using macrophage cell line RAW 264.7 cells. NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds used induced indirect damage through inflammation, and Ni(3)S(2) also showed direct oxidative DNA damage through H(2)O(2) formation. This double action may explain relatively high carcinogenic risk of Ni(3)S(2). 相似文献
5.
Tang Lei Reiter Russel J. Li Zhong-Ren Ortiz Genaro G. Yu Byung Pal Garcia Joaquin J. 《Molecular and cellular biochemistry》1998,178(1-2):299-303
In the present study, the effect of melatonin on oxidative DNA damage induced by kainic acid (KA) treatment was investigated. 8-hydroxy-deoxyguanosine (8-OH-dG) is a main product of oxidatively damaged DNA and was used as the endpoint in these studies. The levels of 8-OH-dG were found to be elevated in the hippocampus and frontal cortex of rats treated with KA. These elevated levels were significantly reduced in animals that were co-treated with melatonin. Thus, there was no difference in 8-OH-dG levels in the brain of control rats compared to those treated with KA (10 mg/kg) plus melatonin (10 mg/kg). The levels of 8-OH-dG also increased in the liver of rats treated with KA. This rise in oxidatively damaged DNA was also prevented by melatonin administration. Melatonin's ability to reduce KA-induced increases in neural and hepatic 8-OH-dG levels presumably relates to its direct free radical scavenging ability and possibly to other antioxidative actions of melatonin. 相似文献
6.
Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes 总被引:1,自引:0,他引:1
Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels. 相似文献
7.
DNA damage levels and biochemical repair capacities associated with XRCC1 deficiency 总被引:2,自引:0,他引:2
Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell extract (WCE) repair assays and DNA damage measurement techniques, we examined systematically the quantitative contribution of XRCC1 to specific biochemical steps of BER and single-strand break repair (SSBR). Our studies reveal that XRCC1-deficient Chinese hamster ovary WCEs exhibit normal base excision activity for 8-oxoguanine (8-OH-dG), 5-hydroxycytosine, ethenoadenine, and uracil lesions. Moreover, XRCC1 mutant EM9 cells possess steady-state levels of endogenous 8-OH-dG base damage similar to those of their wild-type counterparts. Abasic site incision activity was found to be normal in XRCC1-deficient cell extracts, as were the levels of abasic sites in isolated chromosomal DNA from mutant cells. While one- and five-nucleotide gap filling was not affected by XRCC1 status, a significant approximately 2-4-fold reduction in nick ligation activity was observed in EM9 WCEs. Our results herein suggest that the primary biochemical defect associated with XRCC1 deficiency is in the ligation step of BER/SSBR, and that XRCC1 plays no significant role in endogenous base damage and abasic site repair, or in promoting the polymerase gap-filling step. 相似文献
8.
《Mutation Research/Reviews in Genetic Toxicology》1994,317(1):25-42
A wide variety of oxidative DNA lesions are commonly present in untreated human and animal DNA. One of these lesions, 8-hydroxydeoxyguanosine, has been shown to lead to base mispairing (mutation) on DNA replication. Other lesions remain to be investigated in this respect. Oxidative DNA lesions on cell replication may, in appropriate circumstances, lead to proto-oncogene activation. Oxidative DNA damage, on fixation, may also lead to cytotoxicity followed by regenerative proliferation. The probable or possible importance of oxidative DNA damage is reviewed for various classes of carcinogens and natural processes, including metal ions, high-energy radiation, miscellaneous chemicals, tumor-promoting agents, polyhydroxyphenols/quinones, lipid metabolism, peroxisome proliferators and thyroid function. It is concluded that although the evidence needs considerable strengthening in many of these examples, the available information indicates the potential importance of oxidative DNA damage in the induction of tumors by these agents. It is also possible that non-cancerous degenerative diseases associated with aging are the result of the accumulation of lesions resulting from unrepaired oxidative DNA damage. 相似文献
9.
Age-related increases of 8-hydroxy-2'-deoxyguanosine and DNA-protein crosslinks in mouse organs 总被引:4,自引:0,他引:4
Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism. 相似文献
10.
Pioglitazone is a prototype of thiazolidinediones, used for the treatment of type 2 diabetes mellitus. Previous studies suggest that pioglitazone might cause DNA damage by generation of oxidative species. In this study, we investigated the mutagenic effects of pioglitazone using sister chromatid exchanges (SCEs), and chromosomal aberrations (CAs) assays in cultured human lymphocytes. In addition, oxidative DNA damage was evaluated in cells culture by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) marker. We also investigated the possible protective effects of vitamin B12, which is associated with DNA repair, on DNA damage induced by pioglitazone. Treatment of the human lymphocytes with pioglitazone (100μM) significantly increases the frequency of SCEs and CAs (p<0.01). In addition, significant elevation in 8-OH-dG release from lymphocytes was observed after treatment with pioglitazone (p<0.01). On the other hand, pretreatment of cultures with vitamin B12 (13.5μg/ml) protected lymphocytes from the genotoxic effect of pioglitazone. Therefore, we conclude that pioglitazone is genotoxic, and it induces chromosomal and oxidative DNA damage in cultured lymphocytes and this toxicity is prevented by pretreatment with vitamin B12. 相似文献
11.
Burkhardt S Reiter RJ Tan DX Hardeland R Cabrera J Karbownik M 《The international journal of biochemistry & cell biology》2001,33(8):775-783
Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers. 相似文献
12.
《Free radical research》2013,47(1-3):23-27
Active oxygen species (AOS) such as O and H2O2 have been shown to be generated from both gas and tar phases of cigarette smoke and it has been suggested that they are involved in carcinogenesis due to cigarette smoking. Therefore, we investigated the effect of cigarette smoking on oxidative DNA damages in human peripheral blood cells using 8-hydroxydeoxy-guanosine (8-OH-dG) as a marker.From ten healthy male volunteers aged 20-22 years, 5 ml of blood was taken before and 10 minutes after smoking 2 cigarettes in 10 minutes. After lysis of blood cell membranes leukocyte DNA was isolated using a DNA extractor and 8-OH-dG levels were determined using high performance liquid chromatography (HPLC) with electrochemical detection.The mean levels of 8-OH-dG increased significantly (P <0.05) from 3.3 ± 0.8/106dG (mean ± SD) to 5.1 ± 2.5 after smoking.These results indicate that cigarette smoking induces oxidative DNA damage in peripheral blood cells in a relatively short time. 相似文献
13.
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) has attracted enormous attention in recent years because it has been acknowledged as a typical biomarker of oxidative DNA damage. In this paper, the electrochemical performance of 8-OH-dG at the poly(3-methylthiophene) (P3MT) modified glassy carbon electrode (GCE) was investigated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The conducting polymer P3MT can effectively decrease the oxidation peak potential of 8-OH-dG and greatly enhance its peak current. In 0.1 M pH 7.0 phosphate buffer solution (PBS), the anodic peak currents of cyclic voltammograms are linear with the 8-OH-dG concentration in two intervals, viz. 0.700-35.0 microM and 35.0-70.0 microM, with the correlative coefficients of 0.9992 and 0.9995, respectively. The detection limit of 8-OH-dG can be estimated to be 0.100 microM (S/N=3). This modified electrode can be used to detect the amount of 8-OH-dG in human urine. Furthermore, the effects of scan rate, pH, and interference of uric acid (UA) for the voltammetric behavior and detection of 8-OH-dG are also discussed. This proposed modified electrode also shows excellent reproducibility and stability that makes it an ideal candidate for amperometric detection of 8-OH-dG in flow injection analysis (FIA) and high performance liquid chromatography (HPLC). 相似文献
14.
C.-S. Yang P.-J. Tsai W.-Y. Chen J.-S. Kuo 《Redox report : communications in free radical research》2013,18(6):379-383
SummaryThe effect of myocardial ischemia and reperfusion on left ventricular interstitial 8-hydroxydeoxyguanosine (8-OH-dG), a possible biomarker for in vivo oxidative deoxyribonucleic acid damage, in anesthetized rats was investigated. A microdialysis probe was implanted. Levels of 8-OH-dG in microdialysates were analyzed via an on-line high performance liquid chromatography system equipped with an electrochemical detector. Myocardial ischemia for 10 or 20 min, induced by clamping of the left anterior descending coronary artery, did not affect 8-OH-dG levels. However, reperfusion following either 10-min or 20-min ischemia significantly increased 8-OH-dG levels in collected microdialysates. Reperfusion-induced increases in 8-OH-dG levels were more prominent in the 20 min ischemia group (as high as 3.5 fold relative to basal levels) than in the 10 min ischemia group as high as 2.0 fold relative to basal levels). In conclusion, we observed that left ventricular interstitial 8-OH-dG concentration increased following myocardial ischemia and reperfusion in anesthetized rats. These results suggest that 8-OH-dG might be a useful biomarker for oxidative damage following myocardial ischemia and reperfusion. 相似文献
15.
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. 相似文献
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. 相似文献
16.
Svoboda P Ko SH Cho B Yoo SH Choi SW Ye SK Kasai H Chung MH 《Analytical biochemistry》2008,383(2):236-242
Using an established high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, fraction collection, and electrochemical detection, the oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH-dG) can be analyzed rapidly and precisely in human urine samples. In addition, by ultraviolet (UV) detection, it was shown recently that it is possible to simultaneously analyze creatinine and 7-methylguanine (m7Gua), an RNA degradation product, in urine. By adding a fluorescence detector to the HPLC system, we now report that it is also possible to detect pteridins such as neopterin and biopterin. The fluorescence detection was evaluated in detail for neopterin, an immune response and tumor marker. The urinary content of neopterin, assessed by using the HPLC method, was verified with a commercial neopterin enzyme-linked immunosorbent assay (ELISA) kit as indicated by the high correlation between the two methods (r = 0.98). In urinary samples from 58 young healthy individuals (male and female nonsmokers, ages 19-39 years), it was found that there was no significant correlation (r = −0.04) between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). In contrast, in urinary samples from 60 old healthy individuals (male and female nonsmokers, ages 60-86 years), there was a significant correlation (r = 0.47) found between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). These findings strongly indicate that the higher level of immune response that was correlating with old age contributes significantly to the higher level of oxidative damage as assessed in the form of 8-OH-dG. Using this type of HPLC system, it is possible to evaluate oxidative DNA damage and immune response simultaneously using the respective urinary markers. These data may contribute to understanding of the pathophysiology of diseases such as infections and tumor progression where both oxidative stress and immune response occur simultaneously. 相似文献
17.
With a recently developed high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, precise fraction collection, and reversed-phase chromatography, the oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OH-dG) was measured in human urine samples. The HPLC analysis was further modified to measure 8-OH-dG in rat and mouse urine samples. In addition, the urinary RNA degradation product 7-methylguanine (m7Gua) was analyzed simultaneously. The correlation coefficient (r) for the correlation between urinary creatinine and m7Gua was 0.9 for rats and 0.8 for humans and mice. Levels of 8-OH-dG in relation to urinary creatinine were compared and found to be similar for humans and rats and twice as high for mice. Urinary levels of m7Gua, as normalized to creatinine, were several-fold higher in rodents as compared with human levels, thereby correlating with the higher resting metabolic rate of rodents. The presented results show that 8-OH-dG and m7Gua can be analyzed simultaneously and reliably in urine from humans and rodents. In addition, m7Gua may be used as a reliable marker instead of creatinine for the normalization of 8-OH-dG in urine from rats and mice and also may be used in addition to normalization with creatinine in measurements of 8-OH-dG in human urine samples. 相似文献
18.
Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells 总被引:1,自引:0,他引:1
Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage. 相似文献
19.
Previous studies have indicated that splenic macrophages migrate into the liver and play a role in endotoxin-induced hepatic damage. The present study was designed to elucidate the mechanisms of hepatocyte injury induced by activated splenic macrophages, focusing especially on endogenously released NO and oxidative DNA alterations in hepatocytes. Splenic macrophages isolated from Wistar rats were incubated with either lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and cocultured with hepatocytes. Nitrite and nitrate levels in the culture medium were measured, and inducible-type NO synthase (iNOS) and nitrotyrosine were determined by immunofluorescence staining. The ratio of 8-hydroxy-deoxyguanosine (8-OH-dG) to deoxyguanosine (dG) was measured by high-performance liquid chromatography, and single-stranded DNA in hepatocytes was detected with acridine orange. NO release and nitrotyrosine expression in hepatocytes increased after 8 h of coculture with activated macrophages, and this coculture also induced increases in the 8-OH-dG/dG ratio and single-stranded DNA in the hepatocytes. These alterations were attenuated by superoxide dismutase (SOD) and NO synthesis inhibitors. A similar pattern of alterations was observed in hepatocytes incubated with SIN-1, and these changes were also prevented by SOD. These results suggest that activated macrophage-derived NO and its oxidative metabolite, peroxynitrite, play key roles in hepatocyte injury during inflammation, and cause subsequent DNA damage in surviving hepatocytes. 相似文献
20.
Ascorbic Acid Inhibits Lipid Peroxidation but Enhances DNA Damage in Rat Liver Nuclei Incubated with Iron Ions 总被引:2,自引:0,他引:2
In this report we studied DNA damage and lipid peroxidation in rat liver nuclei incubated with iron ions for up to 2 hrs in order to examine whether nuclear DNA damage was dependent on membrane lipid peroxidation. Lipid peroxidation was measured as thio-barbituric acid-reactive substances (TBARS) and DNA damage was measured as 8-OH-deoxyguanosine (8-OH-dG). We showed that Fe(II) induced nuclear lipid peroxidation dose-dependently but only the highest concentration (1.0 mM) used induced appreciable 8-OH-dG. Fe(II1) up to 1 mM induced minimal lipid peroxidation and negligible amounts of 8-OH-dG. Ascorbic acid enhanced Fe(II)-induced lipid peroxidation at a ratio to Fe(II) of 1:l but strongly inhibited peroxidation at ratios of 2.5:l and 5:l. By contrast, ascorbate markedly enhanced DNA damage at all ratios tested and in a concentration-dependent manner. The nuclear DNA damage induced by 1 niM FeSO4/5 mM ascorbic acid was largely inhibited by iron chelators and by dimethylsulphoxide and manni-tol, indicating the involvement of OH. Hydrogen peroxide and superoxide anions were also involved, as DNA damage was partially inhibited by catalase and, to a lesser extent, by superoxide dismutase. The chain-breaking antioxidants butylated hydroxytoluene and diphenylamine (an alkoxyl radical scavenger) did not inhibit DNA damage. Hence, this study demonstrated that ascorbic acid enhanced Fe(II)-induced DNA base modification which was not dependent on lipid peroxidation in rat liver nuclei. 相似文献