Molecular mechanisms of DNA damage by PAHs

PAHs 损伤 DNA 的分子机制

• 批准号: 8104007
• 负责人: Ian Alexander Blair
• 金额: $ 29.25万
• 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/8104007
• 关键词: 2' -deoxyadenosine; 4-oxo-2-nonenal; 8-Oxo-2' -Deoxyguanosine; A/J Mouse; Address; Adenine; Affect; Anabolism; Animal Model; Appearance; 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; Deoxyadenosines; 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; Glycols; 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; Oxidoreductase; Parents; Pathway interactions; Peroxidases; Pharmaceutical Preparations; Phase; 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; Smoking; Specificity; Staging; Techniques; Testing; Therapeutic; Tobacco smoke; Transdisciplinary Tobacco Research Center; Transferase; Translational Research; Up-Regulation; Urine; adduct; base; benzo(a)pyrene-DNA adduct; carcinogenesis; detoxication; exhaust; exposed human population; human subject; inhibitor/antagonist; insight; ionization; mouse model; multiple reaction monitoring; non-smoking; novel; novel strategies; nucleocytoplasmic transport; oxidation; oxidized lipid; perhydroxyl radical; programs; protective effect; 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）：多环芳烃DNA损伤的分子机制。多环芳烃（PAHs）可以通过三种主要途径被激活为能够诱导DNA损伤的活性中间体。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，这主要产生稳定的(+)-抗“ans-N2-2”-脱氧鸟苷（dGuo）加合物。其次，CYP过氧化物酶可以将母体多环芳烃转化为自由基阳离子，形成去纯化的N7-和c8 -鸟嘌呤（Gua）和N7-腺嘌呤（Ade）加合物。第三，akr可以将反式二氢二醇转化为邻醌，并通过氧化还原循环生成活性氧（ROS）。ROS可以直接修饰DNA形成7,8-二氢-8-氧-2'-脱氧鸟苷（8-氧- dguo），并诱导脂质氢过氧化物的形成。脂质氢过氧化物经过均解分解形成双功能亲电试剂4-氧-2-壬烯醛，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，探讨多环芳烃环境暴露在诱导氧化应激和dna加合物形成中的作用。我们建立了一种稳定同位素稀释液相色谱-多重反应监测/质谱（LC-MRM/MS）方法，用于特异性分析(+)-anf/- trans - b [a] p -7,8,9,10-四氢-7,8-二醇-9,10-环氧化物- n2 - dguo -加合物，并定量了该加合物在四种不同的人肺源性细胞系中的形成。这些研究意外地揭示了P450s 1A1和1B1的上调对DNA损伤具有保护作用。我们现在提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方法最近也显示，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黑果。这些研究将扩展到B[a] p暴露小鼠模型，其中B[a] p衍生的尿液代谢物和dna加合物将与尿脂氧化产物和黑果一起被量化。转化研究将通过分析尿液B[a]P代谢物来监测人群对B[a]P的暴露情况。这种暴露的影响将通过监测尿B[a] p - dna加合物、黑果和氧化脂质来评估。我们提出以下假设：(1)P450 1A1和1B1通过II期酶促进B[a]P代谢物的去除来保护DNA免受损伤。(2)转运进入细胞核是DNA损伤的重要决定因素。(3)氧化脂质和B[a]P代谢产物的分析将明确氧化应激与B[a]P暴露之间的关系。(4)对尿中B[a]P-和脂质氧化衍生的DNA加合物进行定量分析，将有助于了解在动物模型和暴露于B[a]P的人群中发生的DNA损伤量。拟议的研究将根据四个具体目标进行。目的1。分析核运输受损细胞模型中脂质氧化和B[a] p来源的dna加合物。目标2。建立稳定同位素稀释lc -电子捕获常压化学电离/MRM/MS和免疫亲和LC-MRM/MS方法，分别用于分析尿液B[a]P代谢物和B[a]P衍生dna加合物。目标3。在暴露于B[a]P的小鼠模型中，定量测定尿氧化脂质、黑果、B[a]P代谢产物和B[a]P来源的dna加合物。目标4。吸烟和不吸烟人群暴露于环境中B[a]P代谢产物和B[a]P来源的dna加合物的定量分析。该研究的成功完成将为确定环境暴露导致的DNA损伤的个体间风险因素提供一种新的方法和方法。