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。     

正常食管上皮和食管鳞状细胞癌组织中丙二醛-DNA加合物含量

目的 研究正常食管上皮和食管癌组织中致癌性丙二醛－DNA加合物（M1－dG)含量,探讨DNA氧化损伤与食管癌发生和发展的关系。方法 所有组织标本均来自食管癌高发区河南林县,32例正常食管上皮标本来源于组织活检,30例食管癌组织标本来源于外科手术切除的食管癌。DNA中M1－dG加合物含量以^32P-后标记方法测定。结果 在全部正常食管上皮和癌组织DNA中均检测到M1－dG加合物,但其含量（中位数）在正常食管上皮中为3．4／10^8核苷酸（范围为1．7／10^8-55.4/10^8核苷酸）,远低于食管癌组织的14.1/10^8核苷酸（范围为1.4/10^8-59.0/10^8核苷酸）,差异有极显著性（P＜0．0001）。该加合物水平与受试者的性别、年龄、吸烟状态以及涉及酒精氧化产生自由基的细胞色素p4502E1基因多态性无明显相关。结论 脂质过氧化产生的丙二醛对DNA的损伤可在食管上皮中累积,在癌组织中达相当高的水平,提示M1－dG加合物可能是导致食管癌发生和发展的重要因素。     

芳烃羟化酶及吸烟与肺癌的关系探讨

为研究芳烃羟化酶及吸烟与肺癌的关系，采用ＡＨＨ直接测定法检查了肺癌患者４０例，其它癌症患者３２例及健康人４５例血液淋巴细胞中ＡＨＨ的诱导力。根据吸烟状况和ＡＨＨ诱导力不同分析患肺癌的相对危险度：（１）肺癌组与对照组相比，不吸烟者中ＡＨＨ高诱导力型发生肺癌的相对危险性升高２倍，吸烟者相对危险性升高４－６倍。（２）肺鳞癌组与对照组相比，不吸烟者中ＡＨＨ高诱导力型的相对危险性升高４倍，吸烟者升高７倍。     

彗星试验检测间接致突变物的DNA损伤作用

目的与方法：用彗星试验检测在体外活化与不活化条例下9种间接致突变物（苯并（a）芘、7，12－二甲基苯并蒽、3－甲基胆蒽、2－氨基蒽、2－氨基芴、4－硝基喹啉－1－氧化物、乙基亚硝基胍、黄曲霉素B1和环磷酰胺）对V79细胞的DNA损伤作用。结果：在活化条件下，8种物质出现DNA损伤作用或损伤作用增强，1种物质（4－硝基喹吉林－1－氧化物）DNA损伤作用减弱。结论：加入体外活化系统，彗星试验可检测间接致突变物的DNA损伤。     

杏仁对吸烟导致DNA氧化损伤的保护作用

背景与目的： 研究美国加州杏仁对吸烟诱导的DNA氧化损伤是否具有保护作用。材料与方法： 选取30个吸烟的志愿者,随机分3组（每组10人）,每天分别给予0, 84和168 g的杏仁,共28 d;试验期间各组对象膳食和吸烟量相同,在实验开始前和试验结束后分别收集新鲜尿液测定柯替宁,采静脉血做彗星实验,收集24 h尿测定8-OH-dG。结果： 彗星实验显示,168 g/d剂量组DNA损伤程度比对照组和试验前均显著降低(P<0.05);8-OH-dG 测定结果显示,84 g / d和168 g / d剂量组的8-OH-dG水平明显低于试验前和对照组(P<0.05)。结论： 食用美国加州杏仁可能具有降低吸烟人群DNA氧化损伤的作用。     

100例吸烟者外周血淋巴细胞微核率的检测

长期吸烟对人体是慢性损害的过程,香烟烟雾中含有多种致癌物和致癌前体物质,对人体DNA的损伤和增加患多种癌症的风险已得到流行病学、病理学、细胞遗传学和分子生物学工作的证明。因此,探索应用短期检测法评价吸烟对人体的遗传毒理学效应,从中寻找敏感人群,这在肿瘤防治中是有积极意义的。本文应用微量外周血浓集的淋巴细胞微核测试法,检测了100例吸烟者的微核率改变,现将结果简报如下: 100例吸烟者选自保健条件较好、生活     

淮安市食管癌病例—对照研究（I）：烟,酒因素的作用

目的 研究淮安市烟,酒因素对食管癌的作用,方法 用１：１全人群病例－对照研究的方法调查淮安市５４９对食管癌病例和对照。结果 在调整了年龄,文化程度,腌制品,水果等因素后,发现吸烟是淮安市食管癌的危险因素,与不吸烟人群相比,男性现仍吸烟者ＯＲ为２．５（９５％ＣＩ：１．６－３．８）男性曾经吸烟者ＯＲ为１．９（９５％ＣＩ：０．９～４．０）,男性患食管癌的危险随着吸烟量的增多,吸烟年限的延长而增加,且有显     

导致吸烟者淋巴细胞染色体损伤的因素

人们普遍认为吸烟可以引起许多健康问题。但是吸烟与生物诱导效应以及吸烟者健康损害的广度之间的因果关系尚未得到充分证实。已知香烟雾由数千种化学物质组成,其中一些物质是诱变剂和/或致癌剂。在生物学活性方面,已表明香烟雾及其凝聚物可以和DNA及蛋白质形成加合物,诱导吸烟者淋巴细胞发生染色体畸变、姐妹染色单体互换和基因突变。作者采用淋巴细胞微核分析法,对67位吸烟者和59位非吸烟对照者进行了染色体畸变率分析。发现吸烟者平均微核率略高于非吸烟者,但差别不显著(P     

浊漳河水体中有机污染物的遗传毒性研究

目的： 评价浊漳河水体有机污染物对人外周血淋巴细胞的遗传毒性。方法：采用单细胞凝胶电泳试验(彗星试验)对浊漳河水体3个不同样点的有机提取物所致的人外周血淋巴细胞的DNA损伤进行研究。结果：在一定剂量范围内,各水样有机提取物均能引起人外周血淋巴细胞不同程度的DNA损伤,并随剂量的增高而加重,与阴性对照组相比,差异均具有统计学意义(P<0.01);以每管100 ml剂量组进行多重比较表明,各采样点水样对人血淋巴细胞DNA损伤程度从大到小依次为暴马、黄碾、石梁。结论：浊漳河水体有机污染物对人外周血淋巴细胞DNA有断裂损伤作用,彗星试验技术的应用有助于水环境监测以及水中有机提取物遗传毒性的研究。     

绞股蓝提取物对衰老大鼠DNA损伤的影响

背景与目的探讨绞股蓝提取物对衰老大鼠DNA氧化及烷化损伤的影响。材料与方法成年大鼠颈背部皮下注射D-半乳糖100mg/kg构建衰老动物模型,同时设注射等量生理盐水的正常对照组。已建模的大鼠分为人工合成饲料中添加不同剂量的绞股蓝提取物(200、800、4000mg/kg饲料)及维生素E、C(VE C)各组,8周后采血获取淋巴细胞,用单细胞凝胶电泳法检测淋巴细胞DNA自发损伤及H2O2诱导的氧化损伤;实验结束前1周留取大鼠24h尿液,通过毛细管电泳法检测大鼠尿中O6-甲基鸟嘌呤(O6-MeG)的含量。结果各组大鼠淋巴细胞DNA自发损伤无明显差异(P>0.05)。分别采用5、10和25μmol/LH2O2氧化时,绞股蓝各组及维生素E、C(VE C)组DNA氧化损伤均明显低于衰老对照组(P<0.05),且绞股蓝800、4000mg/kg组DNA氧化损伤与正常对照组及VE C组无显著性差别。衰老对照组尿中DNA烷化损伤代谢产物O-6MeG含量较其他各组偏高,但差别无统计学意义。结论D-半乳糖诱导衰老的大鼠模型DNA抗氧化损伤的能力降低,而DNA烷化损伤无明显改变;在本实验条件下,绞股蓝提取物可有效降低H2O2诱导的DNA氧化损伤,对DNA烷化损伤没有明显作用。     

Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index.

Oxidative DNA damage may be implicated in ageing, carcinogenesis and other degenerative diseases. Oxidative DNA damage can be assessed in humans in vivo from the urinary excretion of the DNA-repair product 8-hydroxydeoxyguanosine (8OHdG). We investigated factors influencing the excretion of 8OHdG in 24 h urine from 83 randomly selected healthy subjects (52 women) aged 40-64 years. For 2 weeks prior to urine collection the subjects kept a weighed diet record. 8OHdG was quantified by an automatic three-dimensional HPLC analysis with electrochemical detection. The 8OHdG excretion was 252 +/- 103 (mean +/- SD) pmol kg body weight/24 h with a range from 78 to 527. Multiple regression analysis identified three factors, smoking, body mass index (BMI) and gender, as significant predictors of the 8OHdG excretion. In 30 smokers the 8OHdG excretion was 320 +/- 99 pmol/kg/24 h opposed to 213 +/- 84 pmol/kg/24 h in 53 non-smokers. According to multiple regression analysis smokers excreted 50% (31-69%; 95% confidence interval) more 8OHdG than non-smokers. In 52 women the 8OHdG excretion was 240 +/- 106 pmol/kg/24 h opposed to 271 +/- 96 pmol/kg/24 h in 31 men. According to the multiple regression analysis men excreted 29% (10-48%) more 8OHdG than women. According to multiple regression analysis the 8OHdG excretion decreased with 4% (2-6%) per increment in BMI measured in kg/m2. The dietary distribution of energy demonstrated no important predictive value with respect to 8OHdG excretion. The intake of the antioxidant vitamins C and E and of vitamin A equivalents, including beta-carotene, was not associated with 8OHdG excretion. The results suggest that smoking increases oxidative DNA damage by approximately 50%. This effect implies potential serious health effects adding to the other well-known health hazards of smoking. The higher 8OHdG excretion in men and lean subjects may be related to a higher rate of metabolism with increased availability of reactive oxygen species. The apparent 7-fold individual variation in oxidative DNA damage carries implications regarding the rate of ageing and the risk of cancer and other degenerative diseases. The excretion of 8OHdG into urine offers a valuable tool for testing such hypotheses in humans.     

Effects of supplementing a low-carotenoid diet with a tomato extract for 2 weeks on endogenous levels of DNA single strand breaks and immune functions in healthy non-smokers and smokers

Increased consumption of tomato products is associated with a decreased risk of cancer. The present study was performed to investigate whether consumption of a tomato oleoresin extract for 2 weeks can affect endogenous levels of DNA single strand breaks in peripheral blood lymphocytes in healthy non-smokers and smokers. We also assessed, the effect of the tomato oleoresin extract on various immunological functions of peripheral blood mononuclear cells. A double-blinded, randomized, placebo-controlled study design was used. Over a period of 2 weeks 15 non-smokers and 12 smokers were given three tomato oleoresin extract capsules daily (each containing 4.88 mg lycopene, 0.48 mg phytoene, 0.44 mg phytofluene and 1.181 mg alpha-tocopherol). The control group received placebos. The baseline level of endogenous DNA damage for non-smokers was slightly (13%) and non-significantly (P = 0.44) lower than that of smokers. Placebo supplementation of non-smokers and smokers for 2 weeks did not significantly affect lycopene plasma levels or DNA damage in either group. Intervention with tomato oleoresin extract resulted in significant increases in total plasma lycopene and resulted in decreased levels of DNA strand breaks of approximately 32 (non-smokers) and 39% (smokers). However, this effect was not statistically significant in either group (P = 0.09 for non-smokers and P = 0.12 for smokers). Analysis of the distribution pattern of DNA strand breaks showed a statistically significant (P < 0.05) increase in the number of comets in class 0 (undamaged) and a decrease in classes 1 and 2 (damaged) after the tomato oleoresin extract intervention in non-smokers. The changes in the smoker group were not statistically significant. Treatment with the tomato extract had no effect on lymphocyte proliferation, NK cell activity, interleukin (IL)-2 production and tumor necrosis factor (TNF)alpha production, but it significantly reduced IL-4 production in smokers (P = 0.009).     

Products of oxidative DNA damage and repair as possible biomarkers of susceptibility to lung cancer

The broad spectrum of oxidative DNA damage biomarkers [urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) and 8-hydroxyguanine (8-OH-Gua)] and the level of oxidative DNA damage and repair in leukocytes DNA were analyzed in three groups of subjects: (a) lung cancer patients [all smokers (n = 51)]; (b) healthy smokers with comparable smoking status (n = 26); and (c) healthy nonsmokers (n = 38). The mean level of 8-OH-Gua in urine samples of 38 healthy nonsmokers reached a value of 1.783 +/- 0.785 nmol/day/kg. This level was significantly lower than that in the urine of the two smoker groups (cancer patients and healthy smokers), in whom the levels reached values of 2.319 +/- 1.271 and 2.824 +/- 0.892 nmol/day/kg, respectively. Urinary excretion of 8-OH-dGuo was similar in all groups of subjects. The level of 8-OH-dGuo in DNA isolated from leukocytes of cancer patients was significantly higher than that in DNA isolated from the group of healthy smokers and nonsmokers (9.44 +/- 4.77 versus 7.20 +/- 2.83 and 5.88 +/- 2.47 molecules/10(6) deoxyguanosine, respectively). Repair activity of 8-OH-Gua, as estimated by the nicking assay, was significantly higher in blood leukocytes of healthy volunteers (44.6 +/- 20.21 and 37.54 +/- 13.43 pmol/h/mg protein for smokers and nonsmokers, respectively) than in the leukocytes of lung cancer patients (24.56 +/- 11.28 pmol/h/mg protein). Because oxidative DNA insult represented by urinary excretion of oxidative DNA lesions was similar in both groups of subjects with similar smoking status, it appears likely that a higher rate of generation of oxidative damage in cellular DNA of lung cancer patients is a result of deficiency of the repair mechanism(s) in this group.     

Generation of 8-hydroxydeoxyguanosine from DNA using rat liver homogenates

In relation to carcinogenesis, aging and other pathologic conditions, urinary 8-hydroxydeoxyguanosine (8OHdG) is widely used as a marker for evaluating the effect of oxidative stress on DNA. Because no reports have described how 8OHdG is generated from DNA in vivo or by biological materials, and how it is excreted into urine, the authors investigated the generation of 8OHdG from DNA, using rat liver homogenate. Oxidatively damaged DNA samples containing different levels of 8OHdG were prepared using ultraviolet irradiation with three different concentrations of riboflavin. Following incubation of damaged DNA samples with rat liver homogenates, the generation of 8OHdG from the DNA was determined using high-performance liquid chromatography with electrochemical detection after ultrafiltration of the incubation mixtures. The generation of 8OHdG was also tested with an anti-8OHdG antibody. The quantity of 8OHdG generated from the DNA by rat liver homogenates was dependent on the 8OHdG levels in the DNA: almost all 8OHdG in the DNA was released as 8OHdG by rat liver homogenates. Generation of 8OHdG correlated with the degradation of DNA. Interestingly, the generated 8OHdG was stable in the presence of rat liver homogenates, whereas deoxyguanosine (dG) rapidly disappeared in the same conditions. Less than 1/10,000 of dG was converted to 8OHdG when dG was incubated with rat liver homogenate. Incubation of 8-hydroxyguanine with rat liver homogenates did not generate 8OHdG. These findings suggest that most of the 8OHdG in DNA is released as 8OHdG during DNA degradation and that, because of its stability, 8OHdG is excreted into urine, thus providing a convenient measure of oxidative damage to DNA.     

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.     

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major metabolite of O-phenylphenol

The generation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymusDNA treated with O-phenylphenol (OPP) or its major metabolites,phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), was studied.The content of 8OHdG residues was increased in DNA treated withPHQ, and the generation of 8OHdG was highly dependent on PHQconcentration. PBQ had little effect on the formation of 8OHdG,and OPP had no effect. The formation of 8OHdG by PHQ was reducedby oxygen radical scavengers such as catalase, sodium benzoateand sodium azide. The PHQ-induced 8OHdG formation was acceleratedby the addition of CuCI or CuCI₂ to the reaction mixture, butwas decreased by the addition of chelating agents such as EDTA,bathocuproinedisulfonic acid disodium salt (bathocuproine disulfonate)and O-phenanthroline. These results demonstrate that hydroxylradicals generated in the process of oxidation of PHQ contributeto the formation of 8OHdG in DNA, and copper ions facilitatethe oxidative DNA damage. Copper ions greatly accelerated thePHQ-induced DNA cleavage in vitro, although they had no effecton cleavage without PHQ. On the other hand, DNA cleavage occurredby the addition of FeCl₂ in the absence and presence of PHQ.FeCI₂ stimulates 8OHdG formation only slightly with or withoutPHQ. Furthermore, the stimulatory effect of FeCl₂ on 8OHdG formationwas observed even in the presence of EDTA. The formation of8OHdG in bladder DNA is likely to be one of a series of eventsleading to bladder tumors seen in rats fed OPP-containing diet.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8- hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)- 1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.      

Increased formation of 8-hydroxydeoxyguanosine, an oxidative DNA damage, in lymphoblasts from Fanconi's anemia patients due to possible catalase deficiency

Fanconi's anemia (FA) cells are highly susceptible to both reactiveoxygen species and mitomycin C (MMC), a DNA cross-linking agent.In this study we have determined the amounts of 8-hydroxydeoxyguanosine(8OHdG), typical of oxidative DNA damage, in Epstein Barr virustransformed lymphoblasts from FA patients and normal controlsby the use of HPLC combined with electrochemical detection.FA cells (HSC72 and 99 cells being assigned to FA complementationgroup A) formed 2–3 times more 8OHdG than control cellsafter incubation with 20 mM H₂O₂ at 37°C for 30 min. FAcells also formed more 8-hydroxyguanosine, typical of oxidativeRNA damage, than control cells. FA cells showed decreased activityto decompose H₂O₂. Although the activity in FA cells was only20–30% less than control cells, the remaining, undecomposedH₂O₂ concentration was almost twice as much in FA cells as incontrol cells, and the remaining H₂O₂ concentration correlatedwell with the amounts of 8OHdG formation. The H₂O₂ decomposingactivity was almost completely inhibited by sodium azide (NaN₃)or aminotriazole, both catalase inhibitors. With these inhibitorsthe amounts of 8OHdG formation were much higher than in thosecells without inhibitors, and were almost the same in controlcells as in FA cells. Catalase activity in FA cell lysates was70–80% of controls. MMC also increased 8OHdG formationin FA cells only at ED₁₀₀ but not at ED₅₀. These results indicatethat FA cells, at least FA complementation group A cells, haveincreased susceptibility to oxidative DNA damage, and that thisincreased susceptibility is possibly due to decreased catalaseactivity. These results also suggest that catalase plays animportant role in protecting DNA from oxidative damage. However,this increased susceptibility to oxidative DNA damage is considerednot to be the major cause of the increased susceptibility toMMC.     

ACCELERATED PAPER: Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells

To clarify the mechanisms of intracellular induction of oxidativeDNA damage, we have investigated the concentrations of intracellularreactive oxygen species and the amounts of 8-hydroxydeoxyguanosine(8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-likecells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). Wedetermined intracellular concentrations of hydrogen peroxideand superoxide by flow cytometry with dichlorofluorescein diacetateand hydroethidine, respectively. We determined the 8OHdG amountswith an electrochemical detector connected to HPLC after anaerobicsample processing. DMSO-HL60 releases superoxide upon stimulationwith phorbol myristate acetate, and the released superoxidedismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with100 nM phorbol myristate acetate increased intracellular hydrogenperoxide, superoxide and 8OHdG (control). Addition of 1000 U/mlcatalase decreased hydrogen peroxide (313% of control) and 8OHdG(20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%)and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG(30.4%), but increased hydrogen peroxide (129.6%). Additionof 200 µM 4-acetamido-4-isothiocyanostilbene-2, 2'-disulfonicacid decreased superoxide (59.9%) and 8OHdG (42.0%). Additionof 0.4% ethanol had no effect on superoxide concentration (102.2%),but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%).Pretreatment of DMSO-HL60 with 0.1 mM FeSO₄ increased 8OHdG(1173%), but decreased hydrogen peroxide(75.8%). These findingsindicate that the extracellularly released superoxide and hydrogenperoxide diffuse into the cell, but that such reactive oxygenspecies are not the direct molecules to induce 8OHdG. Our resultssuggest that 8OHdG is induced by the hydroxyl radical whichis generated from intracellular hydrogen peroxide and superoxide-reducedFe.