Molecular mechanisms of DNA damage by PAHs

PAHs 损伤 DNA 的分子机制

• 批准号: 7302444
• 负责人: Ian Alexander Blair
• 金额: $ 41.94万
• 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/7302444
• 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 DNA损伤的分子机制。多环芳烃（PAHS）可以激活到能够通过三个主要途径诱导DNA损伤的反应性中间体。 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和C8-瓜氨酸（GUA）和N7-腺嘌呤（ADE）加合物。脂质氢过氧化物的形成。 PAH模型探讨了环境暴露于PAHS在诱导氧化应激和DNA添加剂形成中的作用。 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.这些研究意外地表明，P450S 1A1和1B1的上调对DNA损伤具有保护作用。现在，我们建议探索这一令人兴奋的新发现的分子基础。免疫亲和力稳定的同位素稀释LC-MRM/MS方法论最近还显示，脂质氧化衍生的hedguo存在于暴露于高水平B [A] P的受试者的尿液中。这些研究将扩展到B [a] P举动的小鼠模型，其中B [A] P衍生的尿尿代谢产物和DNA-ADDUCT将与尿脂质氧化产物和Hedguo一起量化。翻译研究将通过通过分析尿B [A] P代谢物的分析来监测人群对B [A] P的暴露。该暴露的影响将通过监测同一受试者中的尿B [A] P-DNA-ADDUCTS，HEDGUO和氧化脂质进行评估。我们建议解决以下假设：（1）P450 1A1和1B1通过促进通过II期酶促进B [A] P代谢产物的去除来防止DNA损伤。 （2）进入细胞核是DNA损伤的重要决定因素。 （3）分析氧化脂质和B [A] P代谢物将定义氧化应激与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添加剂的定量分析将洞悉动物模型和暴露于B [A] p的人群中发生的DNA损伤量。拟议的研究将以四个特定目的进行。目的1。分析核转运受损的细胞模型中的脂质氧化和B [A] P衍生的DNA添加剂。目的2。为了开发稳定的同位素稀释LC-电子捕获大气压化学电离/MRM/MS和免疫亲和力LC-MRM/MS方法，用于分析尿液B [A] P代谢物和B [A] P衍生的DNA- adducts。目标3。在暴露于B [a] p的小鼠模型中，要量化尿氧化的脂质，Hedguo，Hedguo，B [A] P代谢产物和B [A] P衍生的DNA- adducts。目的4。用于量化尿液氧化的脂质，hedguo，b [a] p代谢产物和B [A] P衍生的DNA添加剂在吸烟和未吸烟的人群中暴露于环境B [a] p [a] p的人群中。拟议的研究的成功完成将允许提供一种新方法和新方法来确定DNA损伤的个体间危险因素，这是由于环境暴露于B [a] p所致。