Molecular mechanisms of DNA damage by PAHs

PAHs 损伤 DNA 的分子机制

• 批准号: 7487483
• 负责人: Ian Alexander Blair
• 金额: $ 40.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487483
• 关键词: 2' -deoxyadenosine; 4-oxo-2-nonenal; 8-Oxo-2' -Deoxyguanosine; A/J Mouse; ARNT gene; Address; Adenine; Affect; Anabolism; Animal Model; Appearance; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Atmospheric Pressure; Benzo(a)pyrene; Binding; Biological; Biological Assay; Biological Markers; CYP1B1 gene; Carcinogens; Cations; Cell Line; Cell Nucleus; Cell Proliferation; Cell model; Cells; Chemicals; Chromatography; Collaborations; Complex; Cytochrome P450; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA lesion; Deoxycytidine; Deoxyguanosine; Electrons; Environment; Environmental Carcinogens; Environmental Exposure; Environmental Pollutants; Enzymes; Epoxide hydrolase; Epoxy Compounds; Event; Excision; Exposure to; Food; Genetic; Genetic Induction; Genomics; Glutathione; Glycol; Guanine; Human; Hydrolysis; Individual; Individual Differences; Inherited; Institutes; Label; Life; Ligands; Lipid Peroxides; Lipids; Lipoxygenase; Liquid Chromatography; Liquid substance; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mammalian Cell; Mass Spectrum Analysis; Mediating; Metabolic Activation; Metabolism; Methodology; Methods; Modeling; Modification; Molecular; Molecular Weight; Monitor; Mutation; Nuclear; Nucleotide Excision Repair; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Oxides; Parents; Pathway interactions; Peroxidase; Peroxidases; Pharmaceutical Preparations; Phase; Polycyclic Hydrocarbons; Polyunsaturated Fatty Acids; Population; Preparation; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Protein Isoforms; Pyrenes; Quinones; Reaction; Reactive Oxygen Species; Relative (related person); Research; Research Design; Research Personnel; Risk Factors; Role; Sensitivity and Specificity; Series; Skin; Small Interfering RNA; Smoke; Smoking; Specificity; Staging; Standards of Weights and Measures; Techniques; Testing; Therapeutic; Thinking; Tobacco smoke; Transcriptional Activation; Transdisciplinary Tobacco Research Center; Transferase; Translational Research; Up-Regulation; Urine; adduct; base; benzoquinone; carbonyl reductase (NADPH); carcinogenesis; cyclooxygenase 1; cyclooxygenase 2; detoxication; exhaust; exposed human population; human subject; inhibitor/antagonist; insight; ionization; mouse model; non-smoking; novel; novel strategies; nucleocytoplasmic transport; oxidation; oxidized lipid; perhydroxyl radical; programs; protective effect; pyrene; repaired; stable isotope; translational medicine; tumorigenesis; urinary

DESCRIPTION (provided by applicant): Molecular mechanisms of DNA damage by PAHs. Polycyclic aromatic hydrocarbons (PAHs) can be a activated to reactive intermediates capable of inducing DNA damage by three major pathways. First, cytochromes P450 (P450) 1A1 and CYP1B1 can convert frans-dihydrodiols to (+)-a/tf/-diol-epoxides, which yield primarily a stable (+)-anft-fr"ans-N2-2'-deoxyguanosine (dGuo) adduct. Second, CYP peroxidase can convert the parent PAH to radical cations that form depurinating N7- and C8-guanine (Gua) and N7-adenine (Ade) adducts. Third, AKRs can convert trans-dihydrodiols to o-quinones, which redox cycle to generate reactive oxygen species (ROS). The ROS can directly modify DNA to form 7,8-dihydro-8-oxo-2'- deoxyguanosine (8-oxo-dGuo) as well as inducing the formation of lipid hydroperoxides. Lipid hydroperoxides undergo homolytic decomposition to form the bifunctional electrophile 4-oxo-2-nonenal, which then covalently modify DNA to form a heptanone-etheno-dGuo (HsdGuo)-adduct. We propose to use the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P) as a model PAH to explore the role of environmental exposure to PAHs in the induction of oxidative stress and formation of DNA-adducts. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for the specific analysis of (+)-anf/-frans-B[a]P-7,8,9,10-tetrahydro-7,8- diol-9,10-epoxide-N2-dGuo-adduct and quantified the formation of this adduct in four different human lung- derived cell lines. These studies have unexpectedly revealed that up-regulation of P450s 1A1 and 1B1 has a protective effect on DNA damage. We now propose to explore the molecular basis of this exciting new finding. Immunoaffinity stable isotope dilution LC-MRM/MS methodology has also recently revealed that lipid oxidation-derived HedGuo is present in the urine of subjects exposed to high levels of B[a]P through smoking. These studies will be extended to a B[a]P-exposed mouse model in which the B[a]P-derived urinary metabolites and DNA-adducts will be quantified together with urinary lipid oxidation products and HedGuo. Translational research will be conducted by monitoring the exposure of human populations to B[a]P through analysis of urinary B[a]P metabolites. The effects of this exposure will be assessed by monitoring urinary B[a]P-DNA-adducts, HedGuo, and oxidized lipids in the same subjects. We propose to address the following hypotheses: (1) P450 1A1 and 1B1 protect against DNA damage by facilitating removal of B[a]P metabolites through phase II enzymes. (2) Transport into the nucleus is an important determinant of DNA damage. (3) Analysis of oxidized lipids and B[a]P metabolites will define the relationship between oxidative stress and B[a]P exposure. (4) Quantitative analysis of urinary B[a]P- and a lipid oxidation-derived DNA-adducts will provide insight into the amount of DNA damage that has occurred in an animal model and in human populations exposed to B[a]P. The proposed research will be conducted under four specific aims. Aim 1. To analyze lipid oxidation- and B[a]P-derived DNA-adducts in cellular models of impaired nuclear transport. Aim 2. To develop stable isotope dilution LC-electron capture atmospheric pressure chemical ionization/MRM/MS and immunoaffinity LC-MRM/MS methodology for analysis of urinary B[a]P metabolites and B[a]P-derived DNA-adducts, respectively. Aim 3. To quantify urinary oxidized lipids, HedGuo, B[a]P metabolites, and B[a]P-derived DNA-adducts in a mouse model exposed to B[a]P. Aim 4. To quantify urinary oxidized lipids, HedGuo, B[a]P metabolites, and B[a]P-derived DNA-adducts in smoking and non-smoking human populations exposed to environmental B[a]P. Successful completion of the proposed research will permit provide a new approach and novel methodology for determining the inter-individual risk factors for DNA damage, which result from environmental exposure to B[a]P.

描述（由申请人提供）：PAHs 损伤 DNA 的分子机制。多环芳烃 (PAH) 可以被激活为活性中间体，能够通过三种主要途径诱导 DNA 损伤。首先，细胞色素 P450 (P450) 1A1 和 CYP1B1 可以将反式二氢二醇转化为 (+)-a/tf/-二醇-环氧化物，从而主要产生稳定的 (+)-anft-fr"ans-N2-2'-脱氧鸟苷(dGuo) 加合物，CYP 过氧化物酶可以将母体 PAH 转化为形成脱嘌呤的自由基阳离子。第三，AKRs可以将反式二氢二醇转化为邻醌，其氧化还原循环产生活性氧（ROS），ROS可以直接修饰DNA。形成 7,8-二氢-8-氧代-2'-脱氧鸟苷 (8-oxo-dGuo) 以及诱导脂质氢过氧化物的形成。氢过氧化物经过均解分解形成双功能亲电子试剂 4-oxo-2-nonenal，然后共价修饰 DNA 形成庚酮-乙烯-dGuo (HsdGuo)-加合物。我们建议使用普遍存在的环境致癌物苯并[a]芘。 B[a]P）作为多环芳烃模型，探讨环境暴露于多环芳烃在诱导氧化应激和形成DNA 加合物。我们开发了稳定同位素稀释液相色谱-多反应监测/质谱（LC-MRM/MS）方法，用于特异性分析（+）-anf/-frans-B[a]P-7,8,9， 10-四氢-7,8-二醇-9,10-环氧化物-N2-dGuo-加合物，并量化了四种不同的人肺来源细胞系中该加合物的形成。这些研究出人意料地揭示了P450s 1A1和1B1的上调对DNA损伤具有保护作用。我们现在建议探索这一令人兴奋的新发现的分子基础。免疫亲和稳定同位素稀释 LC-MRM/MS 方法最近还揭示，通过吸烟暴露于高水平 B[a]P 的受试者的尿液中存在脂质氧化衍生的 HedGuo。这些研究将扩展到接触 B[a]P 的小鼠模型，其中 B[a]P 衍生的尿液代谢物和 DNA 加合物将与尿液脂质氧化产物和 HedGuo 一起进行定量。转化研究将通过分析尿液苯并[a]芘代谢物来监测人群接触苯并[a]芘的情况。将通过监测同一受试者的尿液 B[a]P-DNA 加合物、HedGuo 和氧化脂质来评估这种暴露的影响。我们建议解决以下假设：(1) P450 1A1 和 1B1 通过 II 相酶促进 B[a]P 代谢物的去除，从而防止 DNA 损伤。 (2) 转运入核是DNA损伤的重要决定因素。 (3) 氧化脂质和 B[a]P 代谢物的分析将确定氧化应激与 B[a]P 暴露之间的关系。 (4) 对尿液 B[a]P 和脂质氧化衍生的 DNA 加合物进行定量分析，将有助于深入了解接触 B[a]P 的动物模型和人群中发生的 DNA 损伤量。拟议的研究将根据四个具体目标进行。目标 1. 在核转运受损的细胞模型中分析脂质氧化和 B[a]P 衍生的 DNA 加合物。目标 2. 开发稳定同位素稀释 LC-电子捕获常压化学电离/MRM/MS 和免疫亲和 LC-MRM/MS 方法来分析尿液 B[a]P 代谢物和 B[a]P 衍生的 DNA 加合物，分别。目标 3. 在暴露于 B[a]P 的小鼠模型中定量尿氧化脂质、HedGuo、B[a]P 代谢物和 B[a]P 衍生的 DNA 加合物。目标 4. 定量暴露于环境 B[a]P 的吸烟和非吸烟人群中尿氧化脂质、HedGuo、B[a]P 代谢物和 B[a]P 衍生的 DNA 加合物。成功完成拟议的研究将为确定因环境暴露于 B[a]P 而导致的 DNA 损伤的个体间风险因素提供一种新的方法和新的方法。