8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage induced by environmental tobacco side-stream smoke and its mechanism

Objective To study the genotoxicity effect of environmental tobacco side-stream smokes (ETSS) on oxidative DNA damage and its molecular mechanism. Methods DNA adduct 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. The level of 8-OHdG in DNA exposed to ETSS was detected by high performance liquid chromatography with electrochemical detection. Organic and inorganic components in ETSS were analyzed by gas chromatography-mass spectrum and atomic absorption spectrum respectively. Results Particle matters (PMs) and volatile organic compounds (VOCs) in ETSS could directly induce oxidative DNA damage and formation of 8-OHdG. There were 123 and 84 kinds of organic components in PMs and VOCs respectively, and 7 kinds of inorganic components in ETSS. Some components, especially quinones and polyphenols in ETSS, could produce free radicals in vitro by auto-oxidation without any biological activity systems, and with the catalytic reaction of metals, the DNA adduct 8-OHdG was produced. Conclusion ETSS have biological oxidative effect on DNA in vitro and in vivo, and expressed direct genotoxicity. 8-OHdG is a valuable biomarker of oxidative DNA damage.     

Oxidative damage to DNA and its relationship with diabetic complications

Objective To detect the oxidative DNA damage in diabetic patients and to investigate the relationship of oxidative DNA damage with diabetes and diabetic nephropathy. Methods Single cell gel electrophoresis （SCGE） was used to detect the DNA strand breaks in peripheral blood lymphocytes, and oxidative DNA damage product and serum 8-OHdG were determined by a competitive ELISA in 47 cases, including 25 patients without diabetic complications, 22 patients with diabetic nephropathy and 25 normal control subjects. Results Diabetic patients showed greater oxidative damage to DNA. The percentage of comet cells and the length of DNA migration （comet tail length） of peripheral blood lymphocytes were significantly increased in patients with diabetes, and significantly higher in patients with diabetic nephropathy than in diabetic patients without vascular complications （P〈0.05）. There was a significant increase in serum 8-OHdG in diabetic patients compared with normal subjects （P〈0.05）. Moreover, serum 8-OHdG was much higher in patients with diabetic nephropathy than in diabetic patients without vascular complications （P〈0.05）. Coneluslon There is severe oxidative DNA damage in diabetic patients. Enhanced oxidative stress may be associated with diabetes, especially in patients with diabetic nephropathy.     

骨关节病软骨细胞DNA氧化性损伤

从１０例临床手术中取得骨关节病标本，包括髋关节置换术的股骨头５例，膝关节骨关节病病灶清除的变性软骨４例，膝关节游离体１例，采用常规方法，坐标不本中提取ＤＮＡ，并通过ＨＰＬＣ－ＥＣ检测ＤＮＡ中的８－羟基－２‘－脱氧鸟苷。结果显示，ＤＮＡ琼脂糖凝胶电泳出现典型细胞程序化死亡特征的梯形改变，且ＤＮＡ氧化性损伤标志８－ＯＨｄＧ的含量增加，提示内源性ＤＮＡ氧化性损伤可能是骨关节病的分子病理机制。     

Expression of 8-hydroxy-2’-deoxyguanosine in gastric carcinomas

Reactive oxygen species may be involved in the progression of gastric carcinomas. To clarify whether the pathology of gastric carcinoma are related to oxidative DNA damage, the expression of 8-hydroxy-2＇-deoxyguanosine （8-OHdG） was examined in 30 patients with gastric carcinomas. Methods： The expression of 8-OHdG and apoptosis in the gastric carcinoma were measured using the methods of immunocytochemistry and deoxynucleartididyl transferase-mediated dUTP-biotin nick end labeling （TUNEL）, respectively. Results： Of the 30 cases, 25（83%） showed stronger immunoreactivity than normal control. The patients with poorly differentiated gastric carcinoma had a larger tumor size and higher labeling indices of TUNEL- and 8-OHdG-positive cells than those with well and moderately differentiated gastric carcinoma. Conclusion： Our findings suggest that oxidative DNA damage is increased in association with necroinflammation in chronic gastric injuries and determination of 8-OHdG is useful in assessing high-grade malignancy in gastric carcinomas.     

抗oh8dG单克隆抗体的制备及生物学特性研究

目的 :制备抗 8-羟基 - 2′-脱氧鸟苷 (oh8d G)的单克隆抗体 ,并通过免疫组化检测胃粘膜中的 oh8d G,评价幽门螺杆菌 (Hp)感染与 DNA氧化损伤的关系。方法 :以 BSA- oh8d G偶联物免疫 BALB/ C小鼠 ,采用杂交瘤技术制备单克隆抗体 ,以竞争性 ELISA、Western blot和免疫组化进行生物学特性鉴定。结果 :建立了两个抗 oh8d G的单克隆抗体杂交瘤细胞株 (ZMC9,ZMD11) ,竞争性 ELISA和 Western blot显示该抗体对 oh8d G有高度特异性 ,免疫组化结果显示 oh8d G在 Hp阳性胃粘膜组织中表达率为 5 5 % ,Hp阴性胃粘膜组织中表达率为 5 % ,两者差异有显著性 (P<0 .0 1)。结论 :本研究制备的单克隆抗体能特异识别 oh8d G;Hp感染可导致胃粘膜组织 oh8d G水平增高。     

An investigation of oxidative DNA damage in pharmacy technicians exposed to antineoplastic drugs in two Chinese hospitals using the urinary 8-OHdG assay

Objective To investigate oxidative DNA damage in pharmacy technicians preparing antineoplastic drugs at the PIVAS (Pharmacy Intravenous Admixture Service) in two Chinese hospitals.Methods Urinary 8-OHdG served as a biomarker.5-Fluorouracil (5-FU) concentrations in air,masks and gloves were determined.The spill exposure of each PIVAS technician to antineoplastic drugs was investigated.Eighty subjects were divided into exposed group Ⅰ,Ⅱ,and control group Ⅰ,Ⅱ.Results 5-FU concentration ratios for gloves and masks in exposed group Ⅰ were significantly higher than those in exposed group Ⅱ (P＜0.05 or P＜0.01).The average urinary 8-OHdG concentrations in exposed group Ⅰ,control group Ⅰ,exposed group Ⅱ,and control group Ⅱ were 14.69±0.93,10.68±1.07,10.57±0.55,and 11.96±0.73 ng/mg Cr,respectively.Urinary 8-OHdG concentration in exposed group Ⅰ was significantly higher than that in control group Ⅰ or that in exposed group Ⅱ (P＜0.01).There was a significant correlation between urinary 8-OHdG concentrations and spill frequencies per technician (P＜0.01).Conclusion There was detectable oxidative DNA damage in PIVAS technicians exposed to antineoplastic drugs.This oxidative DNA damage may be associated with their spill exposure experience and contamination of their personal protective equipment.     

Influence of acupuncture on cognitive function and markers of oxidative DNA damage in patients with vascular dementia

OBJECTIVE:To test the influence of acupuncture on cognitive function and a marker of oxidative DNA damage in patients with vascular dementia(VD).METHODS:Sixteen VD patients were evaluated before and after acupuncture,using the Folstein Mini-Mental State Examination-Revised(MMSE-R) to assess cognitive function,and the ADL-R scale to assess independence in activities of daily living(ADL).Life quality was evaluated using the DEMQOL(Dementia quality of life questionnaire) questionnaire,and syndromes and expression of vascular dementia were evaluated with the Scale for the Differentiation of Syndromes of Vascular Dementia(SDSVD).In addition,the urine concentration of 8-hydroxy-2’-deoxyguanosine(8-OHdG)-a marker of oxidative damage-was quantified with enzyme-linked immunosorbent assay.RESULTS:The MMSE-R and DEMQOL scores were higher after acupuncture than before(P<0.05),while there were no obvious differences in the ADL-R or SDSVD scores(P>0.05).The 8-OHdG content in urine significantly decreased after acupuncture(P<0.05).CONCLUSION:Acupuncture reduces the levels of 8-OHdG and improves cognitive function and quality of life in VD patients,suggesting that acupuncture is beneficial at least in part by preventing oxidative damage.     

褪黑素对丙烯酰胺致大鼠睾丸细胞DNA损伤的拮抗作用

目的研究丙烯酰胺(AA)致大鼠睾丸细胞DNA损伤及褪黑素(MT)对损伤的拮抗作用。方法 40只8周龄SD雄鼠,随机分为空白对照组、MT组、AA组和MT+AA组,每组10只。MT(10 mg.kg-1.d-1)腹腔注射,AA(25 mg.kg-1.d-1)经口灌胃染毒,共30 d。单细胞凝胶电泳检测睾丸细胞DNA损伤(CASP软件测定慧星尾长、慧尾DNA百分含量、尾矩及Olive尾矩),TUNEL标记凋亡细胞,ELISA试剂盒检测睾丸组织中8-羟基脱氧鸟苷(8-OHdG)含量,硫代巴比妥酸法测定丙二醛(MDA)水平,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活力。结果与对照组比较,AA组大鼠睾丸细胞彗星尾长、彗尾DNA百分含量、尾矩、Olive尾矩均显著增加,凋亡细胞增多,睾丸组织8-OHdG和MDA含量增加,SOD活力降低,差异均有统计学意义(均P<0.05)。与AA组比较,MT+AA组大鼠睾丸细胞DNA损伤程度减轻,凋亡细胞数减少,睾丸组织8-OHdG和MDA含量降低,SOD活力升高,差异均有统计学意义(均P<0.05)。结论 AA能够引起大鼠睾丸细胞DNA损伤并诱发凋亡,且对睾丸产生氧化损伤;MT则能拮抗AA引起的损伤。氧化损伤可能是AA引起大鼠睾丸细胞DNA损伤的机制之一。     

2型糖尿病人群DNA氧化损伤的研究

目的了解健康人群尿样中8-羟基脱氧鸟苷（8-OHdG）含量的正常范围。探索尿样中8-0HdG含量水平能否作为反映2型糖尿病患者DNA氧化损伤程度的直接指标。方法采用酶联免疫吸附法（EUSA）检测健康人群和2型糖尿病人群干预前后尿样中8-OHdG的含量。结果2型糖尿病无并发症病例和2型糖尿病伴肾病病例尿样中的8-OHdG含量分别为（22．37±5．62）ng／mgCr和（23．80±7．58）ns／msCr,明显高于健康人的（15．74±6．10）ng／mgCr（P〈0．05）。且2型糖尿病伴肾病病例尿样中的8-OHdG含量要高于无并发症病例,各组病例干预后的尿样中8-OHdG含量均低于干预前的水平。结论2型糖尿病无并发症病例和2型糖尿病伴肾病病例的DNA氧化损伤水平均高于健康人群。规范化的治疗、正确的饮食和运动指导,能有效降低2型糖尿病患者DNA氧化损伤的程度。检测尿样中8-OHdG含量,对于预防、监测和控制2型耱尿病患者病情的发展,都是一个简单而有效的方法。     

特发性弱精子症患者精子DNA氧化损伤

目的:探讨特发性弱精子症患者精浆的氧化应激及其精子DNA的氧化损伤。方法:选择2010年12月至2011年3月就诊于中南大学湘雅医院生殖中心门诊的不孕不育夫妇。以28名特发性弱精子症患者为实验组,选取24名精液分析正常且有生育史的男性为对照组,收集新鲜精液。应用鲁米诺化学发光法检测原始精浆活性氧(reactive oxygen species,ROS)水平;酶联免疫分析法检测精子DNA氧化产物8-羟基脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8-OHdG)浓度。结果:1) 实验组精浆ROS水平增高,与对照组比较差异有统计学意义(P<0.01);两组前向运动精子活动率与精浆ROS水平呈负相关(r=-0.72,P<0.01)。2) 实验组精子8-OHdG浓度高于对照组,差异有统计学意义(P<0.01);两组前向运动精子活动率与精子8-OHdG浓度也呈负相关(r=-0.73,P<0.01)。3) 两组患者精浆ROS水平与精子8-OHdG浓度呈正相关(r=0.77,P<0.01)。结论:特发性弱精子症患者的精子具有DNA氧化损伤,可能与氧化应激有关。过量活性氧产生可能是特发性弱精子症患者精子活力低下的原因之一。     

oh8dG在胃癌组织中的表达及其临床意义

目的研究胃癌组织及正常胃黏膜组织中8-羟-2-脱氧鸟苷(oh8dG)表达,探讨DNA氧化损伤与胃癌发生和发展的关系。方法以免疫组化方法检测40例胃癌组织中(胃癌组)和20例正常胃黏膜组织标本(对照组)中oh8dG的表达。结果 oh8dG在胃癌组和对照组的表达水平有显著性差异,(P<0.05)。胃癌组中oh8dG表达与癌组织浸润深度、淋巴结转移、远处器官转移、临床病理分期有显著相关。结论 oh8dG在胃癌组织中表达水平增高,提示DNA氧化损伤是胃癌发生的重要因素,oh8dG可作为判定胃癌预后的指标。     

Expression of 8-hydroxy-2′-deoxyguanosine in gastric carcinomas

ObjectiveReactive oxygen species may be involved in the progression of gastric carcinomas. To clarify whether the pathology of gastric carcinoma are related to oxidative DNA damage, the expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG) was examined in 30 patients with gastric carcinomas.MethodsThe expression of 8-OHdG and apoptosis in the gastric carcinoma were measured using the methods of immunocytochemistry and deoxynucleartididyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), respectively.ResultsOf the 30 cases, 25 (83%) showed stronger immunoreactivity than normal control. The patients with poorly differentiated gastric carcinoma had a larger tumor size and higher labeling indices of TUNEL- and 8-OHdG-positive cells than those with well and moderately differentiated gastric carcinoma.ConclusionOur findings suggest that oxidative DNA damage is increased in association with necroinflammation in chronic gastric injuries and determination of 8-OHdG is useful in assessing high-grade malignancy in gastric carcinomas.     

血清8-羟基脱氧鸟苷水平升高与癌症相关性抑郁的临床意义

目的DNA氧化损伤标记物8-羟基脱氧鸟苷（8-OH-dG）分别与癌症和抑郁存在密切联系,本研究对该标记物与癌症相关性抑郁间的关系进行了探讨,分析其在癌症抑郁中诊断及评定程度中的临床意义.方法经病理确诊并使用17项汉密尔顿抑郁量表（HAMD）调查评定出的34例无抑郁的癌症患者（无抑郁组）,抑郁患者53例,采用酶联免疫吸附法（ELISA）检测血清8-OH-dG水平.结果抑郁组和无抑郁组的平均8-OH-dG水平分别是（8.73±1.98）ng/mL和（5.44±0.69）ng/mL（t=-11.054,P〈0.01）.8-OH-dG水平与抑郁评分呈正相关（r=0.776）,与是否是少数民族呈负相关（r=-0.214）.逐步Logistic回归分析显示8-OH-dG（OR=7.196）是独立危险因素.结论检测外周血8-OH-dG水平可能有助于癌症相关性抑郁的早期诊断及病情判定.     

Exposure to static magnetic field of pregnant rats induces hepatic GSH elevation but not oxidative DNA damage in liver and kidney

BACKGROUND: The present investigation was designed to evaluate the effects of subacute exposure to static magnetic field (SMF) on some parameters indicative of oxidative stress and on oxidative DNA damage in pregnant rat. METHODS: Females rats (n = 6) were exposed to a SMF (128 mT; 1 h/day) from day 6 to day 19 of pregnancy and were allowed to deliver normally. The control group (n = 6) was not exposed to SMF. Dams were sacrificed 3 days after delivery. The effects of subacute exposure to SMF on oxidative states were assessed on the measurements of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and catalase (CAT). The level of 8-oxo-dG was measured using high-performance liquid chromatography coupled to electrochemical detection. RESULTS: Subacute exposure to SMF failed to alter plasma GPx, MDA, CAT and SOD respectively in liver and kidney. By contrast, SMF increased total GSH (+56%, p <0.05) and reduced GSH (+108%, p <0.05) in liver. Our results showed that the exposure to SMF did not induce oxidative DNA lesions in liver and kidney. CONCLUSIONS: The data do not provide evidence that subacute SMF exposure causes DNA damage in liver and kidney in pregnant rats. The present results suggest that hepatic GSH plays an important role in protection against SMF during pregnancy. These changes in antioxidant status (GSH) lead to some adaptive responses due to activation of systems controlling the body oxidative mechanism balance.     

慢性砷暴露对小鼠肾组织DNA损伤的研究

目的:通过检测8-羟基脱氧鸟嘌呤(8-OHdG)在砷暴露小鼠肾脏组织中的表达和分布,探讨砷的肾毒性作用机制。方法:昆明种小鼠40只,随机分为4组,每组10只,分别为低剂量组(1ppmAs2O3)、中剂量组(2ppm As2O3)、高剂量组(4ppm As2O3)和生理盐水对照组,连续染毒60天后断头处死,用HE染色法进行组织病理学观察,用免疫组化及图像分析方法检测肾组织内8-OHdG的水平。结果:与对照组相比,1ppm、2ppm和4ppm染砷组小鼠肾组织出现不同程度细胞肿胀,胞浆空泡变性,核固缩、溶解及血管扩张、充血等病理学改变,近曲小管较肾小球损伤严重。免疫组化结果表明,肾组织中8-OHdG含量明显增高,并呈现明显的剂量-反应关系,其总光密度值与对照组比较具有显著性差异(P<0.01)。而且,8-OHdG在近曲小管的表达明显高于肾小球区域。结论:慢性低剂量砷暴露可引起小鼠肾组织DNA的氧化损伤和病理变化,尤其对肾脏近曲小管的损伤作用较为明显。     

The Effects of HBx Gene on the Expression of DNA Repair Enzymes hOGG1 and hMYHα mRNA in HepG2 Cells

To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHa and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the β-actin as the interior control, real-time polymerase chain reaction （Real-time qPCR） was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector （HepG2/pDNA3.1）. The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx （0.021±0.007） was significantly lower than that of HepG2 （0.099±0.041） （P〈0.05） and HepG2/pDNA3.1 （0.121±0.005） （P〈0.05）. However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains （P〉0.05）. The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 （P〈0.05）. It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.     

HPLC-MS/MS法研究普萘洛尔对映体在不同种属肝微粒体中的代谢差异

目的 采用高效液相色谱-串联质谱法（HPLC-MS/MS）建立R-（+）-普萘洛尔和S-（-）-普萘洛尔在肝微粒体孵育体系中的含量测定方法,并分别比较普萘洛尔不同对映体在大鼠、犬、猴以及人4种肝微粒体中的代谢特征。方法 分别将R-（+）-普萘洛尔和S-（-）-普萘洛尔溶解在由烟酰胺腺嘌呤二核苷酸磷酸（NADPH）启动孵育的不同种属的肝微粒体中,在孵育不同的时间后加入乙腈终止反应。使用电喷雾离子源（ESI）,以卡维地洛为内标,在多反应监测模式（MRM）下进行正离子扫描,分别测定各孵育体系中R-（+）-普萘洛尔和S-（-）-普萘洛尔的浓度。以孵育0 min时不同构型的普萘洛尔的质量浓度为参照,分别计算R-（+）-普萘洛尔和S-（-）-普萘洛尔在不同肝微粒体样品中的药物剩余百分比和体外代谢半衰期和固有清除率。结果 普萘洛尔的线性范围为0.05～10.00μg/mL,定量下限为0.05μg/mL;日内、日间精密度的RSD%均小于13%。在4个种属的肝微粒体孵育体系中,R-（+）-普萘洛尔在大鼠肝微粒体中代谢快,在猴肝微粒体中次之,在犬和人肝微粒体中代谢慢,体外消除半衰期依次为大鼠<猴&l...     

Eales病患者血液中氧化及抗氧化水平分析

目的： 通过对不同时期Eales病患者血液中氧化及抗氧化水平的比较分析,探讨氧化损伤与Eales病发病的关联性。方法：Eales病首发病例、治愈病例各10例为研究对象,同时选择健康男性献血队员10例为正常对照。以8-羟基脱氧鸟苷（8-OHdG）作为DNA氧化损伤的标记物,以巴比妥酸盐反应物质（TBARS）作为脂质过氧化的标记物,应用气相色谱质谱联用仪（GC/MS）及TBARS检测试剂盒测定不同时期Eales病患者血液中8-OHdG及丙二醛（MDA）的含量。应用相应的测定试剂盒分别对研究对象血清中总抗氧化能力、总SOD活力、GSH-PX活力及维生素E含量进行测定。结果：不同时期Eales病患者血液中8-OHdG及MDA含量均显著 高于健康对照组（P<0.001）;首发病例组血液中8-OHdG及MDA含量显著高于治愈病例组（P<0.001）。不同时期Eales病患者血清中总抗氧化能力、抗氧化酶活性及维生素E含量等抗氧化指标均较正常对照组显著下降（P<0.05）。另外,治愈病例组血清中抗氧化指标虽然较首发病例组有所升高,但仍显著低于正常对照组（P<0.05）。结论：氧化应激与Eales病的发病具有一定的关联性。推测补充维生素C、E等抗氧化物质可能对Eales病的治疗和预防起重要的作用。     

DNA氧化损伤模型的构建

目的探索Fenton反应(Fe2+H2O2→Fe3++     

维生素E对苯并[a]芘诱导雄性大鼠的生殖毒性的保护作用

目的：探讨维生素E(vitamin E,VE)对苯并[a]芘(Benzo(a)pryene,B[a]P)诱导的雄性大鼠的生殖毒性的保护作用及其机制。方法：将60只28 d龄70~90 g Sprague Dawley (SD)大鼠随机分为空白、溶剂对照组、B[a]P组(5 mg/kg)及低(10 mg/kg)、中(50 mg/kg)、高(100 mg/kg)剂量VE联合B[a]P处理组共6组,每组各10只;连续灌胃30 d并检测各组精子质量、睾丸组织中超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、8-羟基脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHdG)和丙二醛(malondialdehyde,MDA)的含量;HE染色观察睾丸形态,采用彗星实验测定细胞DNA损伤程度。结果：与对照组相比,B[a]P组精子计数明显降低、精子畸形率、8-OHdG及MDA含量明显升高,SOD和GSH-Px活性显著下降(P<0.05);VE联合组SD大鼠的睾丸组织损伤程度明显减轻;上述各项氧化应激和DNA损伤指标也均明显改善;且随着VE剂量的增加,保护效应呈现明显的剂量-反应关系,差异均具有统计学意义(P<0.05)。结论：VE对B[a]P诱导的SD雄性大鼠生殖损害具有保护作用。