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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

基本信息

批准号：
9039079
负责人：
Thomas E Spratt
金额：
$ 33.88万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-20 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9039079
关键词：
2&apos -Deoxythymidine Aerodigestive Tract 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 glycosylase DNA-Directed DNA Polymerase Deoxyguanosine Embryo Escherichia coli Etiology 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 Son of Sevenless Proteins Time Tobacco Tobacco smoke Tobacco smoking Tobacco-Related Carcinoma Transcription-Coupled Repair United States Work adduct base carcinogenesis cigarette smoking genotoxicity in vivo innovation insight kidney cell methylpurine mortality novel repaired research study

项目摘要

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率的单一主要原因，并且是许多癌症（包括肺癌、上呼吸消化道癌、膀胱癌和胰腺癌）的风险因素。烟草烟雾中最强的致癌物之一是4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）。NNK被生物活化为有效的亲电体，其反应形成甲基和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经得到很好的表征，并且目前的范例是甲基- DNA加合物在烟草诱导的癌症的病因学中比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。我们的初步研究结果表明，O2-POB-dT在人类细胞中修复效率低下，并在SOS诱导的E.大肠杆菌和哺乳动物细胞。本申请的目的是确定O2-POB-dT形成突变并在哺乳动物细胞中修复的机制。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的准确和诱变旁路的聚合酶和（2）确定POB加合物修复的机制。旁路中涉及的聚合酶将在细胞中测定，其中特定聚合酶被siRNA下调，并且在体外用纯化的聚合酶测定。将用无细胞提取物检查跨损伤合成过程中链转换的作用。将通过离体和体外实验的组合来评价修复机制。NER和BER的作用将使用修复蛋白缺陷的细胞进行离体评估，使用HPLC-MS/MS测定法测量DNA加合物的水平。O2-POB-dT通过NER和BER的修复将使用合成的寡脱氧核苷酸在体外检查。转录偶联NER对O 6-POB-dG和O2- POB-dT的作用将用一种新的修饰的宿主细胞再活化来探测。