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

作为评价抗氧化剂在癌症化学预防中安全性的一种方法，我们用人培养细胞和~（32）P标记的人p53抑癌基因和c-Ha-ras-1原癌基因的DNA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. A损伤，用抗氧化剂处理人培养细胞，并用脉冲场凝胶电泳法检测。结果呢。用维生素A、视黄醇、α-生育酚和槲皮素处理检测细胞DNA损伤。此外。我们用HPLC-ECD定量测定了8-羟基脱氧鸟苷（8-OH-dG）的形成，这是DNA氧化损伤的指标。为了检测对分离的DNA. A的损伤，在金属离子存在下，将抗氧化剂与32 P标记的DNA片段孵育，并获得放射自显影。α-生育酚、维生素A、视黄醛、槲皮素和N-乙酰半胱氨酸在Cm（II）存在下引起位点特异性DN，A-损伤。过氧化氢酶和浴铜灵。Cu（I）特异性螯合剂。在Cu（Ⅱ）存在下，DNA断裂发生在胸腺嘧啶和胞嘧啶残基上。所谓的“抗氧化剂”在某些情况下起抗氧化剂的作用，但在另一些情况下也起促氧化剂的作用。换句话说，抗氧化剂在某些情况下具有损伤DNA的能力，尽管抗氧化剂在其他情况下保护免受氧化应激。这些氧化性DNA损伤可能是导致和/或促进肿瘤发生多阶段的原因。在推荐抗氧化剂用于癌症化学预防之前，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.