大剂量维生素E和C抗氧化活性及对DNA损伤影响的研究

背景与目的:观察大剂量维生素E(VitaminE,VE)和C(VitaminC,VC)的抗氧化活性及对大鼠DNA氧化损伤、烷化损伤的影响。材料与方法:将72只Wistar大鼠(雌雄各半)随机分为6组,每组12只,即对照组(基础饲料,VE、VC摄入量分别为5、0mg/kg·d),VC组(VE5mg/kg·d、VC1000mg/kg·d),VE1组(VE33mg/kg·d、VC0mg/kg·d),VE2组(VE500mg/kg·d、VC0mg/kg·d),VE1 VC组(VE33mg/kg·d、VC1000mg/kg·d)和VE2 VC组(VE500mg/kg·d、VC1000mg/kg·d),实验期为8周。实验结束前收集动物24h尿液,实验结束后处死动物,留取血液,分离血浆并收集淋巴细胞,测定超氧化物歧化酶(Superoxidedismutase,SOD)、谷胱甘肽过氧化物酶(Glutathioneperoxidase,GSH-Px)活性、丙二醛(Malondialdehyde,MDA)含量,分析DNA损伤。结果:VE1组的抗氧化活性显著提高:血浆SOD、GSH-Px活力明显高于对照组(P<0.05)、MDA含量低于对照组;而VE1 VC组SOD、GSH-Px则较VE1组显著下降(P<0.05)。VE1组10μmol/LH2O2诱导的淋巴细胞DNA氧化损伤为150.42AU,显著低于其它组(P<0.05);该组DNA烷化损伤产物尿O6-甲基鸟嘌呤(O6-methylguanine,O-6meG)含量为0.89mg/g肌酐,比对照组、VE2组分别下降了50.28%、50.00%(P<0.05)。结论:较大剂量VE能提高大鼠抗氧化活性,降低DNA氧化损伤及烷化损伤,但联合补充大剂量VC时可能对其影响产生拮抗作用;过大剂量VE和VC补充时均未观察到有利作用,并且可能降低机体的遗传稳定性。     

吸烟引起人体氧化损伤的可能机制

背景与目的： 通过多项生物标志物研究吸烟对DNA氧化损伤、脂质过氧化和氧化防御机制的影响。 材料与方法： 选取年龄在18～25岁的男性吸烟志愿者60名和非吸烟者30名,分为吸烟组和非吸烟组。分别通过高效液相色谱-电化学检测法(HPLC-ECD)测定24 h尿样中8-羟基脱氧鸟苷(8-OHdG)水平;应用彗星实验测定外周血淋巴细胞DNA链的断裂情况;采用高效液相色谱-紫外线检测法(HPLC-UV)测定24 h尿样中丙二醛(MDA)、丙酮(ACON)和戊醛(PP)水平;采静脉血测定血浆超氧化物歧化酶(SOD)、谷胱苷肽过氧化氢酶(GPX)和过氧化氢酶含量。 结果： 吸烟组尿8-OHdG和外周血淋巴细胞DNA的断裂分别比非吸烟组高185％和97 ％(P均<0.01),尿MDA、ACON和PP较非吸烟组显著增高(P均<0.01),SOD、GPX和过氧化氢酶分别较非吸烟组低15％,10％和9％(P≤0.01)。 结论： 吸烟可以引起机体的氧负荷,造成DNA氧化损伤、脂质过氧化和抗氧化酶的改变。     

三氯生的体外遗传毒性评价

目的：应用体外碱性彗星试验、体外胞质分裂阻滞微核细胞组学试验和细菌回复突变试验（Ames试验）评价三氯生（TCS）的体外遗传毒性。方法：采用TK6人淋巴母细胞进行体外彗星试验和体外微核试验,共设定5个剂量（3.5、8.8、17.5、26.3和35 μmol/L）组。同时,应用鼠伤寒沙门氏菌TA98、TA100和DNA修复酶缺陷型YG7108（Ogt^-/Ada^-）菌株对TCS进行Ames试验,共设定8个剂量（0.000 5、0.001 67、0.005、0.016 7、0.05、0.167、0.5和1.67 μg/皿）组。结果：与对照组比较,彗星试验结果显示,TCS在各个剂量下均能引起TK6细胞的尾长、尾部DNA强度和尾矩的增加,差异均具有统计学意义（P均 < 0.01）,且尾部DNA强度呈现剂量效应（r=0.943,P=0.017）;微核试验表明,TCS未引起TK6细胞微核率升高（P > 0.05）;Ames试验结果表明TCS未引起TA98、TA100和YG7108菌株的回复突变菌落数增加（P > 0.05）。结论：TCS主要引起细胞DNA损伤,但未对TK6细胞造成染色体损伤,对Ames试验菌株不具有致突变作用。     

氧化/还原因子ref-1在肺癌组织中的细胞定位表达及与8-OH-dG的关系

目的氧化/还原因子-1(ref-1),也称为AP核酸内切酶(APE),是氧化还原和DNA碱基切除修复途径中的重要一员.本研究旨在探讨ref-1在肺癌中的表达及其与氧化损伤标志8-羟基-脱氧鸟苷(8-OH-dG)的关系. 方法用免疫组化法检测150例肺癌组织、120例癌旁肺组织、40例肺良性病变和40例正常肺组织中氧化/还原因子ref-1基因的蛋白质表达;分析有关暴露因素对ref-1表达的影响,探讨ref-1基因与8-OH-dG的关系. 结果肺癌和正常肺组织皆有ref-1的表达,但ref-1在细胞内的定位有所不同,70.0%(105/150)的肺癌组织ref-1从通常的胞核定位移位到胞浆,仅7.5%(3/40)的正常肺组织有ref-1移位胞浆的现象,两者差异显著,P<0.01.未发现肺癌患者的性别、年龄、吸烟和肿瘤家族史等因素与ref-1细胞定位之间有联系.ref-1移位胞浆与肺组织中8-OH-dG的含量呈显著正相关,Spearman相关系数为0.702,P<0.01. 结论 ref-1的细胞定位对其氧化/还原和修复功能有着重要影响,可作为监测细胞DNA修复功能的指标.     

对PTEN缺失小鼠成纤维细胞中DNA氧化损伤的研究

背景与目的:研究FIEN缺失细胞中活性氧(ROS)水平、DNA氧化损伤和抗氧化能力的变化.材料与方法:运用化学/荧光发光分析、免疫细胞化学、中性单细胞凝胶电泳技术,分别比较Pten / MEFs与Pten-/-MEFs细胞中ROS、8-羟基脱氧鸟苷(8-OH-dG)水平、DNA双链断裂(DSBs)以及抗氧化能力的差异.结果:Pten-/-MEFs细胞中ROS、8-OH-dG水平明显增高,DSBs增加,抗氧化能力减弱.结论:PTEN可能通过调控细胞内ROS水平拮抗DNA氧化损伤.     

三丁基锡引起人羊膜细胞氧化损伤及DNA损伤的研究

背景与目的： 研究三丁基锡 (tributyltin,TBT)对人羊膜细胞FL (human amnion cells) 氧化损伤和DNA损伤的诱导作用。 材料与方法： 将不同浓度TBT (0、2、4、6、8、10 μmol/L),分别对FL染毒2 h和4 h,各染毒组同时设不加TBT的对照组,染毒后分别用MTT法检测TBT对FL细胞增殖率的影响,用DCFH_DA法检测FL细胞活性氧自由基 (ROS)水平,用彗星实验检测TBT对FL细胞DNA的损伤。 结果： TBT对FL细胞染毒4 h时,其2、8、10 μmol/L浓度组的细胞增殖率较对照组显著下降(P<0.05,P<0.01,P<0.001),且随TBT浓度升高而增殖率呈下降的趋势。TBT 3、4 μmol/L染毒组FL细胞的ROS水平较对照组升高,且4 μmol/L染毒组与对照组比较差异具有统计学意义(P<0.05)。在TBT 2、3、4 μmol/L染毒组随着TBT浓度的升高,FL细胞核尾长、尾相均显著升高(P均<0.05)。 结论： TBT可引起FL细胞的氧化损伤及DNA损伤。     

胸水细胞8-OH-dG、k-ras和p53基因的表达及其临床意义

背景与目的:DNA的氧化损伤是外来致癌物导致癌变发生的重要机制之一。8-羟基脱氧鸟嘌呤(8-hydoxy-2deoxyguanosine,8-OH-dG)作为DNA氧化损伤的指标,在肺癌的发生、发展及预后扮演着重要角色。研究发现肺组织癌变的过程中8-OH-dG表达水平与k-ras、p53基因突变的关系密切,故作为肺癌标记物在良、恶性判别诊断中具有重要意义。本课题拟探讨胸水细胞8-OH-dG、k-ras和p53基因表达与肺癌的关系,并分析它们在肺癌的微创性良、恶性判别诊断中的价值。方法:用免疫细胞化学法测定53例肺癌患者、53例非肺癌患者的胸水细胞中8-OH-dG、k-ras和p53基因的蛋白表达情况。结果:肺癌组、非肺癌组胸水细胞8-OH-dG阳性率分别为75.5%(40/53)与15.1%(8/53),k-ras基因的蛋白表达阳性率分别为64.2%(34)与3.8%(2),p53基因的蛋白表达阳性率分别为69.8%(37/53)与18.9%(10/53),P值均<0.01,两组比较差异有高度显著性;53例肺癌患者胸水细胞8-OH-dG、k-ras和p53基因的蛋白表达分值均数分别为1.68±1.21、1.32±1.06与1.57±1.15,经等级相关分析8-OH-dG与k-ras基因的蛋白表达呈高度正相关(RS=0.643,P<0.01),8-OH-dG和p53基因的蛋白表达呈高度正相关(RS=0.827,P<0.01),k-ras和p53基因的蛋白表达呈高度正相关(RS=0.897,P<0.01)。结论:肺癌     

运用单细胞凝胶电泳研究稀土元素钬对小鼠骨髓细胞DNA的损伤

背景与目的:探讨稀土元素钬对小鼠骨髓细胞DNA的损伤.材料与方法:给小鼠灌胃氧化钬的盐酸溶液,24h后取股骨骨髓细胞进行单细胞凝胶电泳,观察有无染色体损伤和DNA损伤.结果:试验表明在20～80 mg/kg剂量范围内,彗星细胞的尾长随剂量的增加而增长,而头长则随剂量的递增而缩短,并且在40～80 mg/kg剂量范围内与阴性组差异存在显著性;同时,拖尾率与DNA损伤率也随剂量的增加而升高.结论:稀土元素钬对小鼠骨髓细胞DNA具有一定的损伤作用.     

吸烟诱发人血淋巴细胞DNA氧化损伤的研究

目的：研究吸烟对人血淋巴细胞DNA氧化损伤的作用。方法：采用高效液相色谱系统结合电化学分析法对吸烟和不吸烟的男性健康人外周血淋巴细胞DNA中氧化损伤的生物标志物－8－羟基－2－脱氧鸟苷（8－OHdG）的含量进行分析。结果：①吸烟者比不吸烟者淋巴细胞DNA中的8－OHdG显著提高（P＜0．05）,且8－OHdG的含量与每天吸烟支数吴正相关。②吸烟对DNA氧化损伤的效应个体差异较大,本测定方法可用于对吸烟高敏感人对吸烟高敏感人群的筛选。结论：吸烟可引起人血淋巴细胞DNA氧化损伤。     

彗星分析技术检测辐射和化学物质诱导的DNA损伤

目的： 用彗星分析技术(comet assay)检测邻苯二胺(o-phenylenediamine, o-PDA)和60 Coγ-射线对CHL细胞DNA的损伤。 方法： CHL细胞经0、 2、 4、 6、 8 μmol / L的邻苯二胺染毒和0、 2、 4、 6、 8、 10、 15 Gy 的60 Co γ-射线照射后,进行单细胞凝胶电泳,测定彗星尾长。采用Origin 4.2软件建立两种诱变剂的剂量与CHL细胞彗星尾长的剂效关系。 结果： DNA的损伤程度(以彗星尾长为指标)随60 Coγ-射线剂量的加大和o-PDA浓度的增加而增强,CHL细胞彗星尾长(TL)与60 Coγ-射线的剂效关系符合线性平方模型：TL=20.41＋2.42D＋0.38D 2;与o-PDA浓度的剂效关系符合线性平方模型：TL=1.90＋1.46C＋0.52C 2。 结论： 60 Co γ-射线剂量和o-PDA的浓度与细胞DNA损伤程度具有剂效关系;彗星分析是一种分析环境危害因子遗传毒性的有效技术。     

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.     

彗星实验检测紫外线诱导的K562细胞DNA损伤

背景与目的研究紫外线(Ultraviolet,UV)诱导K562细胞DNA损伤情况,评价彗星实验改良方法及分析参数的可靠性。材料与方法采用0.3mW/cm2UV以用不同时间(3、5、10、40、60、120、180、240s)照射K562细胞,诱导细胞DNA损伤,用彗星实验检测,CASP软件分析。结果尾长、彗星长、尾矩、Olive尾矩4个参数与紫外线照射时间有良好的时间依赖关系。结论0.3mW/cm2的紫外线照射K562细胞3s即可造成细胞DNA损伤,CASP分析软件可以用于彗星检测分析,其中尾长、彗星长、尾矩、Olive尾矩可作为彗星实验的分析指标,所改良的彗星实验系统可靠性较强,且基本上解决了实验中常见的脱胶现象,采图更方便。     

宫颈癌放疗患者外周血淋巴细胞DNA损伤的研究

目的：评价宫颈癌放疗患者外周血淋巴细胞的DNA损伤。方法：取15例宫颈鳞癌放疗患者放疗前及放疗累积剂量为10、20、30、40、50、60Gy时的外周静脉血,以彗星实验检测淋巴细胞的DNA损伤。结果：各累积剂量组淋巴细胞DNA拖尾率比照射前显著升高（P〈0.01）,并且在0～60Gy之间,拖尾率与累积照射剂量之间成线性关系,回归方程为y=12.133＋0.230x,相关系数r=0.964（P〈0.05）。各累积剂量组尾长比照射前均增加（P〈0.01）,在照射剂量累积30Gy时,尾长达最大。之后随照射累积剂量的进一步增加,尾长稍有下降,但是与照射前相比仍持续在较高的水平（P〈0.01）,尾长与累积剂量间不成线性关系。结论：宫颈癌患者放疗后可造成外周血淋巴细胞DNA损伤。     

Cigarette smoking induces an increase in oxidative DNA damage, 8- hydroxydeoxyguanosine, in a central site of the human lung

To evaluate the effect of cigarette smoking on oxidative stress in lung tissues, we compared the levels of a type of oxidative DNA damage, 8- hydroxydeoxyguanosine (8-OH-dG), in tissue obtained from the central sites of lungs from 14 current smokers, seven ex-smokers and nine non- smokers. The mean level of 8-OH-dG in the lung tissues from smokers was 1.43-fold higher than that of the non-smokers (the difference was statistically significant, P = 0.0262). A positive correlation between the 8-OH-dG levels in normal lung tissues and the Brinkman index was obtained from smokers and ex-smokers (r = 0.525; P = 0.0134). A positive correlation was also obtained for the 8-OH-dG levels in normal lung tissues and the number of cigarettes smoked per day (r = 0.436; P = 0.0132). These results suggest that oxidative DNA damage is induced in lung DNA by cigarette smoking.      

Relationship between DNA damage and total antioxidant capacity in patients with glioblastoma multiforme

AimsTo assess oxidative DNA damage and total antioxidant capacity (TAC) in glioblastoma multiforme (GBM) and to compare the results with normal brain tissues.Materials and methodsOxidative DNA damage and TAC were evaluated in GBM tissues extracted from 26 patients and in normal brain tissues of 15 subjects who underwent autopsy within the first 4 h of death. Oxidative DNA damage was assessed by measuring 8-hydroxy-2-deoxyguanosine (8-OH-dG) using the 8-OH-dG enzyme immunoassay kit, a quantitative assay for 8-OH-dG, and TAC was analysed using the ImAnOx colorimetric test system for the determination of antioxidative capacity. The results were compared between two groups and any correlation between 8-OH-dG and TAC was sought.ResultsThe median level of TAC in GBM (121.5 nmol/g wet tissue) was remarkably lower than that in normal brain tissue (298 nmol/g wet tissue). The difference was statistically significant (P = 0.00001). In contrast, oxidative DNA damage was significantly higher in patients with GBM (74.9 ng/g wet tissue) than in controls (34.71 ng/g wet tissue). Again, the difference was statistically significant (P = 0.00001). We also found a negative correlation between oxidative DNA damage and TAC (P < 0.001).ConclusionsThese findings indicate that the degree of oxidative DNA damage is increased and TAC is decreased in GBM. Oxidative DNA damage is correlated with the levels of TAC.     

Genetic instability in bladder cancer assessed by the comet assay

BACKGROUND: Latent genetic instability has been associated with an increased risk for several cancers. We used the comet assay (single-cell gel electrophoresis) to assess whether genetic instability, as reflected by susceptibility to DNA damage, was associated with the risk of bladder cancer in a case-control study. METHODS: We used the comet assay to measure baseline and benzo[a]pyrene diol epoxide (BPDE)- and gamma-radiation-induced DNA damage in individual peripheral blood lymphocytes from 114 incident case patients with bladder cancer and 145 matched healthy control subjects. All subjects provided personal information, including smoking history. DNA damage was visualized with the comet assay and quantified by the Olive tail moment parameter, a relative measure. Multivariable analysis was used to assess relative risks for bladder cancer associated with DNA damage. All statistical tests were two-sided. RESULTS: Baseline levels of DNA damage were statistically significantly higher in case patients (tail moment = 1.40) than in control subjects (tail moment = 1.21) (difference = 0.19, 95% confidence interval [CI] = 0.04 to 0.32; P =.015), as were gamma-radiation-induced (tail moment = 4.76 versus 4.22; difference = 0.54, 95% CI = 0.11 to 0.96; P =.013) and BPDE-induced (tail moment = 4.06 versus 3.45; difference = 0.61, 95% CI = 0.23 to 0.99; P =.002) DNA damage. When data were dichotomized at the median value for DNA damage in control subjects and adjusted for age, sex, ethnicity, and smoking status, an increased estimated relative risk of bladder cancer was statistically significantly associated with DNA damage at baseline (odds ratio [OR] = 1.84, 95% CI = 1.07 to 3.15) and after gamma-radiation (OR = 1.81, 95% CI = 1.04 to 3.14) but not after BPDE treatment (OR = 1.69, 95% CI = 0.98 to 2.93). CONCLUSION: Latent genetic instability as measured by the comet assay is associated with an increased estimated relative risk of bladder cancer.     

Gamma-radiation-induced single cell DNA damage as a measure of susceptibility to lung cancer: a preliminary report

The comet assay is a sensitive and rapid method for detecting DNA single-strand and double-strand breaks and the individual cell's DNA repair profile. This pilot study was designed to determine whether the comet assay could measure inherited susceptibility to lung cancer. We applied the comet assay in the alkaline condition to test the DNA damage in gamma-irradiated and untreated cultured peripheral blood lymphocytes of 31 cases with previously untreated lung cancer and 39 controls. For each culture, 200 consecutive cells were examined and the number of cells with DNA uncoiling under electrophoresis () was recorded. The comet tail length (the radius of the nucleus plus the length of the migrated DNA) at 400-fold magnification was measured for the first 50 identified comet cells. The mean number of induced comet cells was significantly higher in cases (96.0+/-45.7) than matched controls (68.9+/-35.8) (P<0.05). However, no significant difference was observed in induced comet tail length between cases and controls. When we categorized the number of comet cells by the 75th percentile value in the controls, a higher number of comet cells was associated with significantly increased risk for lung cancer [odds ratio = 4.8 (confidence intervals of 1.5, 15.2)] after adjustment for age, sex, ethnicity and smoking status. The number of gamma-irradiation-induced comet cells (r=0.499, P<0.05) and comet tail length (r=0.520, P<0.05) correlated with the results on a previously reported lung cancer susceptibility marker, bleomycin sensitivity. Also, the number of gamma-irradiation-induced comet cells correlated with the results of the benzo[alpha]pyrene diol epoxide mutagen sensitivity assay, which quantifies induced chromatid breaks (r=0.275, P<0.05). The comet assay might be a simple and inexpensive tool for detecting genetic susceptibility to lung cancer.     

Preliminary evaluation of DNA damage related with the smoking habit measured by the comet assay in whole blood cells.

The alkaline single-cell gel electrophoresis (SCGE) assay, also called the comet assay, is a rapid and simple method for the detection of DNA damage in individual cells. The objective of this study was to establish if the alkaline SCGE assay in whole blood cells gives similar results as the same method in isolated lymphocytes, because whole blood cells are simpler and more economical to use, specifically in human genotoxic biomonitoring. To validate the method, we first used mouse blood cells, because mouse is one of the most commonly used animals in genetic toxicology testing. Groups of seven CF1 male mice were given i.p. injections of relatively low doses of methyl methanesulfonate (25 mg/kg body weight), a direct acting genotoxic agent, or cyclophosphamide (50 mg/kg body weight), which requires metabolic activation. Three, 6, 8, 12, 16, 20, and 65 hours after treatment, 5 microL of blood were collected from each animal and were processed for the alkaline SCGE assay. On the basis of an analysis of tail moment, the results showed that this assay can detect DNA damage induced by both kinds of alkylating mutagens. We then did a preliminary study to assess the status of DNA damage in a young (19 to 23 years old) healthy population of male smokers (n = 6) and nonsmokers (n = 6) using the comet assay in whole blood cells. A significant difference was observed between the two groups, showing that the method is able to detect DNA damage in the smoking group despite the short time that the volunteers had actually been smoking.