Genotoxicity and Repair of Tobacco-Specific Nitrosamine DNA Adducts

烟草特异性亚硝胺 DNA 加合物的基因毒性和修复

• 批准号: 8514612
• 负责人: Thomas E Spratt
• 金额: $ 33.26万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-20 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514612
• 关键词: 2' -Deoxythymidine; Affect; Base Excision Repairs; Base Pairing; Biological; Biological Assay; Biological Markers; Bladder; Butanones; Bypass; Cancer Etiology; Carcinogens; Cells; Chemicals; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA-3-methyladenine glycosidase II; DNA-Directed DNA Polymerase; Deoxyguanosine; Embryo; Escherichia coli; Etiology; Gastrointestinal tract structure; Goals; High Pressure Liquid Chromatography; Human; In Vitro; Individual; Ions; Knowledge; Lead; Lung; Malignant Neoplasms; Mammalian Cell; Measures; Methylation; Minor Groove; Modeling; Molecular; Mutagenesis; Mutation; Nitrosamines; Nucleotide Excision Repair; Pancreas; Pathway interactions; Play; Polymerase; Predisposition; Property; Proteins; Pyrimidine; Research; Resistance; Risk Factors; Rodent; Role; Small Interfering RNA; Smoking; Son of Sevenless Proteins; Time; Tobacco; Tobacco smoke; Transcription-Coupled Repair; United States; Work; adduct; base; carcinogenesis; cigarette smoking; genotoxicity; in vivo; innovation; insight; kidney cell; mortality; novel; repaired; research study

DESCRIPTION (provided by applicant): Tobacco-smoking is the single major cause of cancer mortality in the US, and is a risk factor for a number of cancers including lung, upper aero-digestive tract, bladder and pancreas. One of the most powerful carcinogens in tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is bioactivated to potent electrophiles that react to form methyl and 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts. Role of methyl-DNA adducts in carcinogenesis have been well characterized and the current paradigm is that methyl- DNA adducts are more important than POB-DNA adducts in the etiology of tobacco-induced cancers. However, recently it was found that O2-POB-dT adduct is the most persistent POB adduct in NNK-treated rodents. Our preliminary results show that O2-POB-dT is inefficiently repaired in human cells and is mutagenic in SOS-induced E. coli and mammalian cells. The objective of this application is to determine the mechanisms by which O2-POB-dT forms mutations and is repaired in mammalian cells. These goals will be examined in two specific aims: (1) to determine the polymerases involved in accurate and mutagenic bypass of O2-POB-dT and (2) to determine the mechanisms by which the POB-adducts are repaired. The polymerases involved in the bypass will be determined in cells in which specific polymerases are down regulated by siRNA, and in vitro with purified polymerases. The role of strand switching during translesion synthesis will be examined with cell- free extracts. The repair mechanisms will be evaluated by a combination of ex vivo and in vitro experiments. The roles of NER and BER will be evaluated ex vivo using cells with deficient repair proteins using a HPLC- MS/MS assay to measure levels of DNA adducts. The repair of O2-POB-dT via NER and BER will examined in vitro using synthetic oligodeoxynucleotides. The role of transcription-coupled NER for O6-POB-dG and O2- POB-dT will be probed with a novel modified host cell reactivation.

描述（由申请人提供）：吸烟是美国癌症死亡的唯一主要原因，并且是多种癌症的危险因素，包括肺癌、上呼吸消化道、膀胱和胰腺。烟草烟雾中最强的致癌物之一是 4-(甲基亚硝基氨基)-1-(3-吡啶基)-1-丁酮 (NNK)。 NNK 被生物激活，可与有效的亲电子试剂发生反应，形成甲基和 4-(3-吡啶基)-4-氧代丁基 (POB) DNA 加合物。甲基-DNA加合物在致癌作用中的作用已得到很好的表征，目前的范式是甲基-DNA加合物在烟草诱发癌症的病因学中比POB-DNA加合物更重要。然而，最近发现 O2-POB-dT 加合物是 NNK 处理的啮齿动物中最持久的 POB 加合物。我们的初步结果表明，O2-POB-dT 在人类细胞中修复效率低下，并且在 SOS 诱导的大肠杆菌和哺乳动物细胞中具有诱变性。本应用的目的是确定 O2-POB-dT 在哺乳动物细胞中形成突变和修复的机制。这些目标将在两个具体目标中进行检查：(1) 确定参与 O2-POB-dT 精确和诱变旁路的聚合酶，(2) 确定 POB 加合物修复的机制。旁路中涉及的聚合酶将在特定聚合酶被 siRNA 下调的细胞中测定，​​并在体外用纯化的聚合酶测定。将用无细胞提取物检查链转换在跨损伤合成过程中的作用。修复机制将通过离体和体外实验相结合的方式进行评估。 NER 和 BER 的作用将使用修复蛋白缺陷的细胞进行离体评估，并使用 HPLC-MS/MS 测定来测量 DNA 加合物的水平。将使用合成寡脱氧核苷酸在体外检查通过 NER 和 BER 对 O2-POB-dT 的修复。转录偶联 NER 对 O6-POB-dG 和 O2-POB-dT 的作用将通过一种新的改良宿主细胞再激活来探测。