Exposure and biological response biomarkers of cigarette smoke

描述（由申请人提供）： 香烟烟雾的暴露和生物反应生物标志物。暴露于烟草烟雾（主流和环境）是美国死亡的主要原因。卷烟烟气是一种极其复杂的混合物，在主流烟气和侧流烟气中含有约3800种成分，其中包括大量的多环芳烃（PAHs）。吸烟者提供了PAH暴露的极端模型，将允许开发暴露和生物反应生物标志物。大量证据表明，多环芳烃是肺癌、皮肤癌和膀胱癌的致病因子。此外，烟草烟雾与氧化应激、胰腺癌、心血管疾病和慢性阻塞性肺病（COPD）有关，尽管多环芳烃的具体作用尚不清楚。有趣的是，侧流烟对心血管的影响几乎和主流烟一样大。本提案源于我们在过去六年中使用稳定同位素方法在蛋白质、脂质和DNA生物标志物定量方面取得的重大进展，以及我们对氧化应激过程中酶调节的基础研究。以前用于分析氧化性DNA损伤的方法充满了许多方法学问题，因此目前的技术水平涉及使用COMET测定来测量8-氧代-2 '-脱氧鸟苷（dGuo）损伤。我们最近设计了一种基于免疫亲和稳定同位素稀释液相色谱-串联质谱（LC-MS/MS）的更定量的方法，可以很容易地详细说明烟草吸烟者的研究。我们还发现，氧化应激可以诱导醛酮还原酶（AKR）的1C家族的形成。AKR 1C 3是一种酶，我们最近发现它负责将前列腺素（PG）D2转化为有效的支气管收缩剂11 p-PGF 2。这提供了氧化应激和COPD之间的额外潜在联系，以及涉及AKR 1C 3抑制的新治疗策略的潜力。最后，初步研究表明，吸烟者的尿液中存在一种只能由脂质过氧化作用产生的DNA加合物，而非吸烟者的尿液中则完全不存在这种加合物。我们建议通过开发暴露和生物反应的体内生物标志物小组来建立这些令人兴奋的新发现，我们假设这将使区分非吸烟者队列和无疾病吸烟者队列成为可能。将通过在以下三个具体目标下进行研究来检验这一假设：目标1。发现B[a]P和B[a]P-7，8-二酮是否诱导NHBE细胞中的AKR 1C/2并增加氧化应激以在DNA中形成8-氧代-dGuo和HedGuo，诱导HASM细胞中的AKR 1C 3并增加强效支气管收缩剂11 p-PGF 2的生物合成，作为PAH暴露的潜在尿液和EEC生物反应生物标志物。目标二：发现用B[a]P及其氧化代谢物处理NHBE和HASM细胞后分泌的蛋白质作为PAH暴露的潜在血清生物学应答生物标志物。目标3：对尿液中的体内暴露和反应生物标志物以及EBC和血清中的生物反应生物标志物进行预测和细化分析，以区分非吸烟者和无疾病的吸烟者。成功完成拟议的研究将提供一组暴露的生物标志物和对烟草烟雾的生物反应将在未来的研究中具有重要的实用性，旨在阐明基因环境相互作用与癌症，心血管疾病和COPD等疾病之间的关系。

DESCRIPTION (provided by applicant): Exposure and biological response biomarkers of cigarette smoke. Exposure to tobacco smoke (mainstream and environmental) is a leading cause of death in the US. Cigarette smoke is an extremely complex mixture, which some 3800 constituents including numerous polycyclic aromatic hydrocarbons (PAHs), in both the mainstream and sidestream (environmental) smoke fractions. Cigarette smokers provide an extreme model of PAH exposure that will permit both exposure and biological response biomarkers to be developed. There is substantial evidence that PAHs are causative agents in lung, skin, and bladder cancer. Furthermore, tobacco smoke is associated with oxidative stress, pancreatic cancer, cardiovascular disease, and chronic obstructive pulmonary disease (COPD), although the specific role of PAHs is not clear. Interestingly, the cardiovascular effects of sidestream smoke are almost as great as mainstream smoke. The present proposal stems from significant advances we have made over the last six years in the quantification of protein, lipid, and DNA biomarkers using stable isotope methodology and our basic research into enzyme regulation during oxidative stress. Previous methods for analyzing oxidative DNA damage have been fraught with numerous methodological problems so that the current state-of-the-art involves the use of a COMET assay to measure 8-oxo-2'-deoxyguanosine (dGuo) lesions. We have recently devised a more quantitative method based on immunoaffinity stable isotope dilution liquid chromatography- tandem mass spectrometry (LC-MS/MS) that can be readily elaborated to studies of tobacco smokers. We also showed that oxidative stress could induce the formation of aldo-keto reductases (AKRs) of the 1C family. AKR1C3 is the enzyme, which we recently showed is responsible for the conversion of prostaglandin (PG) D2 to the potent bronchoconstrictor 11p-PGF2. This provides an additional potential link between oxidative stress and COPD as well as the potential for a new therapeutic strategy, which involves AKR1C3 inhibition. Finally, preliminary studies have revealed that a DNA-adduct than can only arise from lipid peroxidation is present in the urine of cigarette smokers but is completely absent in urine from non-smokers. We propose to build on these exciting new findings by developing panels of in vivo biomarkers of exposure and biological response, which we hypothesize will make it possible to distinguish a cohort of non-smokers from a cohort of disease-free tobacco smokers. The hypothesis will be tested by conducting research under the following three specific aims: Aim 1. To discover whether B[a]P and B[a]P-7,8-dione induce AKR1C/2 in NHBE cells and increase oxidative stress to form 8-oxo-dGuo and HedGuo in DNA, induce AKR1C3 in HASM cells and increase the biosynthesis of the potent bronchoconstrictor 11p-PGF2, as potential urine and EEC biological response biomarkers of PAH exposure. Aim 2: To discover secreted proteins following treatment of NHBE and HASM cells with B[a]P and its oxidative metabolites as potential serum biological response biomarkers of PAH exposure. Aim 3: To conduct predictive and refinement analyses of in vivo exposure and response biomarkers in urine together with biological response biomarkers in EBC and serum in order to distinguish non-smokers from disease-free tobacco smokers. Successful completion of the proposed research will provide a panel of biomarkers of exposure and biological response to tobacco smoke will have significant utility in future studies designed to elucidate the relationship between gene environment interactions and diseases such as cancer, cardiovascular disease, and COPD.