Analysis of the human DNA damageby antioxidants for evaluation of safety in cancer chemoprevention

作为一种评价抗癌化学预防安全性的方法，我们利用人培养细胞和~(32)P标记的人抑癌基因和c-Ha-ras-L癌基因片段，研究了抗氧化剂是否具有损伤人DNA的能力。为了检测细胞DNA的损伤，用抗氧化剂处理培养的细胞，并用脉冲场GET电泳法进行检测。结果就是。用维生素A、视黄醇、α-生育酚和槲皮素处理细胞DNA损伤。更重要的是。我们用高效液相色谱-电子捕获技术检测了氧化损伤的指标8-羟基脱氧鸟苷(8-OH-DG)的形成。在维生素A、视黄醇和N-乙酰半胱氨酸处理的细胞中，8-羟基脱氧鸟苷(8-OH-DG)的形成显著增加。为了检测分离的DN.A的损伤，将抗氧化剂与~(32)P标记的DNA片段在金属离子存在下孵育，得到放射自显影。α-生育酚、维生素A、视黄醇、槲皮素和N-乙酰半胱氨酸在Cm(II)存在下引起部位特异性的DN，A-损伤。过氧化氢酶和巴托普林。一种铜(I)特异性的络合物。抑制Co(II)介导的DNA损伤，提示H_2O_2和Curl的参与)。在铜(II)存在的情况下，在胸腺嘧啶和胞嘧啶残基上观察到DNA裂解的频率。因此，所谓的抗氧化剂在某些情况下作为抗氧化剂，在其他情况下也作为促氧化剂。换句话说，抗氧化剂在某些情况下具有破坏DNA的能力，尽管在其他情况下抗氧化剂可以防止氧化应激。这些氧化DNA损伤可能是多阶段癌变的始动和/或促进肿瘤发生的原因。在建议使用抗氧化剂治疗癌症化疗前，应评估其安全性和有效性。

As a method to evaluatesafety in cancer chemoprevention, we have investigated whether antioxidants have the ability of the human DNA damage, using human cultured cells and 32P-labeledisolated DNA fragnrents of the human p53 tumor suppressor gene and c-Ha-ras-l prctooncogene. For detection of ce1lularDN.A damage, human cultured celles were treated with antioxidants, and examined by the pulsed-field get electrophoresis method. As the result. celularDNA damage was detected by the treatment of vitamin A, retinal, alpha-tocopherol and quercetin. Furthermore. we measured 8-hydroxy deoxyguanosine (8-OH-dG) formation, which was an index to an oxidative DNA lesion, quaruitativelyby HPLC-ECD.lntracellu1ar8-OH-dG formation significantly increased in cells treated with vitamin A, retinal and N-acetylcysteine. For detection of damage to isolated DN.A, antioxidants were incubated with 32P-labled DNA fragment in the presence of metalions, and the autoradiogram was obtained. alpha-Tocopherol, vitamin A, retinal, quercetin and N-acetylcysceine caused site-specific DN,A-damage in the presence of Cm(II). Catalase and bathocuproine. a Cu(I)-specific chelaror. inhibited the Co(II)-mediated DNA damage, suggesting the involvement of H_2O_2 and Curl).The DNA cleavage was observed frequency at thymine and cytosine residues in the presence of Cu(II).So-called "antioxidants" act as antioxidants in some circumstances, but also act as prooxidants in other circumstances. In another word, antioxidants become to have ability of damaging DNA in some cases, although antioxidants protect from oxidative stress in other cases. These oxidative DNA damage could be responsible for initiation and/or tumor promotion the multi-stage of carcinogenesis. In is requested chat safety and efficacy should be estimated before recommending use of antioxidants for cancer chemopreventton.