Molecular mechanisms of DNA damage by PAHs

描述（由申请人提供）：多环芳烃对DNA损伤的分子机制。多环芳烃（PAHs）是一种活性中间体，通过三种主要途径诱导DNA损伤。首先，细胞色素P450（P450）1A 1和CYP 1B 1可以将反式-二氢二醇转化为（+）-α/β-二醇-环氧化物，其主要产生稳定的（+）-反式-反式-N2 - 2 ′-脱氧鸟苷（dGuo）加合物。第二，过氧化物酶可以将母体PAH转化为自由基阳离子，形成脱嘌呤的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，其氧化还原循环产生活性氧物质（ROS）。ROS可直接修饰DNA形成7，8-二氢-8-氧代-2 '-脱氧鸟苷（8-oxo-dGuo），并诱导脂质过氧化氢的形成。脂质氢过氧化物发生均裂分解，形成双功能亲电试剂4-氧代-2-壬烯醛，然后共价修饰DNA，形成庚酮-乙烯基-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）作为一个模型多环芳烃，探讨环境暴露的多环芳烃在诱导氧化应激和DNA加合物的形成的作用。我们开发了一种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/-反式-B [a]P-7，8，9，10-四氢-7，8-二醇-9，10-环氧-N2-dGuo-加合物，并定量了该加合物在四种不同的人肺衍生细胞系中的形成。这些研究意外地揭示了P450 1A 1和1B 1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方法最近也揭示了脂质氧化衍生的HedGuo存在于通过吸烟暴露于高水平B[a]P的受试者的尿液中。这些研究将扩展至B[a] P暴露小鼠模型，其中将对B[a] P衍生的尿液代谢物和DNA加合物以及尿液脂质氧化产物和HedGuo进行定量。将通过分析尿液B[a]P代谢物监测人群对B[a]P的暴露，从而进行转化研究。将通过监测相同受试者的尿B[a]P-DNA加合物、HedGuo和氧化脂质来评估该暴露的影响。我们提出了以下假设：（1）P450 1A 1和1B 1通过促进II相酶清除B[a]P代谢物来保护DNA免受损伤。(2)进入细胞核的转运是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和B[a]P暴露之间的关系。(4)定量分析尿B[a]P和a脂质氧化衍生的DNA加合物将提供深入了解的DNA损伤的数量，发生在动物模型和暴露于B[a] P的人群中。目标1.分析核转运受损细胞模型中脂质氧化和B[a] P衍生的DNA加合物。目标2.建立稳定同位素稀释LC-电子捕获大气压化学电离/MRM/MS和免疫亲和LC-MRM/MS方法，分别用于分析尿中B[a]P代谢物和B[a] P衍生的DNA加合物。目标3.为了定量暴露于B[a] P的小鼠模型中的尿氧化脂质、HedGuo、B [a]P代谢物和B[a] P衍生的DNA加合物。为了量化暴露于环境B[a]P的吸烟和非吸烟人群中的尿氧化脂质、HedGuo、B [a]P代谢物和B[a] P衍生的DNA加合物。拟议研究的成功完成将允许提供一种新的方法和新的方法来确定DNA损伤的个体间风险因素，这是由环境暴露于B[a] P引起的。

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.