BINDING OF AROMATIC HYDROCARBONS TO NUCLEIC ACIDS

芳香烃与核酸的结合

• 批准号: 3166725
• 负责人: ELEANOR G ROGAN
• 金额: $ 12.19万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3166725
• 关键词: DNA; NAD(H) phosphate; adduct; benzopyrenes; biotransformation; chemical binding; chemical carcinogen; chemical carcinogenesis; chemical synthesis; cysteine; cytochrome P450; electrochemistry; epoxides; exonuclease; fluorescence spectrometry; hybridomas; hydrogen peroxide; laboratory mouse; laboratory rat; lipid peroxides; liver metabolism; microsomes; molecular site; monoclonal antibody; nuclear magnetic resonance spectroscopy; nucleic acid structure; oxidation; peroxides

Most chemica1 carcinogens require metabolic activation to intermediates which react with DNA to form adducts and perhaps initiate cancer. Carcinogenic polycyclic aromatic hydrocarbons (PAH) are metabolized by two main pathways, monooxygenation and one-electron oxidation, forming reactive diol-epoxides or radical cations, respectively, as ultimate intermediates. The objectives of this grant are to continue elucidating the benzo(a)pyrene (BP)- DNA adducts formed by one-electron oxidation, to begin investigating their significance, and to examine the hypothesis that one mechanism by which cytochrome P-450 activates PAH is cooxidation in reactions supported by hydroperoxides formed by lipid peroxidation in the cell. To accomplish this we will 1) Identify BP-DNA adducts formed chemical1y and enzymically in vitro and in topically-treated mice and mammary-treated rats using monoclonal antibodies raised against BP-deoxyguanosine adducts synthesized electrochemically. 2) Examine the stable and labile DNA adducts formed by reaction of BP with a 229 base-pair fragment of pBR322 DNA by a) analyzing single-strand breaks in DNA reacted with BP radical cation perchlorate or treated with BP in the horseradish peroxidase system, b) analyzing alkali-labile 1esions in the BP-treated DNAs, and c) digesting BP-treated DNAs, using exonuclease III to identify the sites of stable adducts. 3) Examine the ability of cytochrome P-450 to cooxidize BP in reactions supported by hydroperoxides of various chain lengths by a) determining and comparing the profiles of BP metabolites and levels of binding of BP to DNA, b) determining the inhibitory effects of trapping agents on the binding of BP to DNA, c) identifying the BP- DNA adducts formed with activation by rat liver microsomal cytochrome P-450 with cumene hydroperoxide, and d) identifying the BP-cysteine adducts formed with hydroperoxidesupported cytochrome P-450. These experiments are expected to demonstrate the presence of both stable and labile BP-DNA addvcts formed by one-electron oxidation in target tissues and identify sites of adduct formation in DNA sequences. In addition, we will determine the ability of bio1ogically relevant hydroperoxides to support cytochrome P-450- mediated activation of BP.

大多数Chemica1致癌物都需要代谢激活 与DNA反应形成加合物的中间体，也许 引发癌症。 致癌多环芳烃 （PAH）由两种主要途径（单二合一）代谢 单电子氧化，形成反应性二醇或自由基 阳离子分别为最终中间体。 目标 这笔赠款是继续阐明苯并（a）pyrene（bp） - 由一电子氧化形成的DNA加合物开始 研究其意义，并检查假设 细胞色素P-450激活PAH的一种机制是 由氢过氧化物支持的反应中的二氧化作用 细胞中的脂质过氧化。 为此，我们将1） 鉴定BP-DNA加合物形成化学作品，并在体外酶促形成 并使用局部治疗的小鼠和乳腺治疗的大鼠 针对BP-脱氧加合物提出的单克隆抗体 通过电化学合成。 2）检查稳定和不稳定 由BP与229碱基对片段反应形成的DNA加合物 通过a）分析DNA反应中的单链断裂的PBR322 DNA 用BP自由基阳离子高氯酸盐或用BP处理 辣根过氧化物酶系统，b）分析碱性 - 法属1SION 在BP处理的DNA中，c）使用BP处理的DNA，使用 外切核酸酶III识别稳定加合物的位置。 3）检查 细胞色素P-450在反应中二氧化bp的能力 由a）由各种链长的氢过氧化物支撑） 确定和比较BP代谢物和水平的曲线 BP与DNA的结合，b）确定的抑制作用 捕获BP与DNA的结合，c）识别BP- 由大鼠肝微粒体激活形成的DNA加合物 用液质过氧化物和d）识别的细胞色素P-450 由氢过氧化物抑制的细胞色素形成的BP半胱氨酸加合物 P-450。 这些实验有望证明存在 由一电子 目标组织中的氧化并识别加合物形成的位点 在DNA序列中。 此外，我们将确定 与生物学相关的氢过氧化物以支持细胞色素P-450- BP的介导的激活。