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。在美国，接触烟草烟雾(主流和环境)是导致死亡的主要原因。卷烟烟雾是一种极其复杂的混合物，主流和侧流(环境)烟雾组分中约有3800种成分，包括大量的多环芳烃(PAHs)。香烟吸烟者提供了一种暴露于多环芳烃的极端模型，这将允许开发暴露和生物反应生物标记物。有大量证据表明，多环芳烃是肺癌、皮肤癌和膀胱癌的病原体。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疾病(COPD)有关，尽管多环芳烃的具体作用尚不清楚。有趣的是，侧流烟雾对心血管的影响几乎与主流烟雾一样大。目前的建议源于我们在过去六年中在使用稳定同位素方法对蛋白质、脂肪和DNA生物标记物进行量化方面取得的重大进展，以及我们对氧化应激过程中的酶调节的基础研究。以前分析DNA氧化损伤的方法存在许多方法学问题，因此目前最先进的方法包括使用彗星分析来测量8-氧-2‘-脱氧鸟苷(Dguo)损伤。我们最近设计了一种基于免疫亲和力稳定同位素稀释液相色谱-串联质谱仪(LC-MS/MS)的更定量的方法，可以很容易地阐述到烟草吸烟者的研究。我们还发现，氧化应激可以诱导1C家族的醛酮还原酶(AKRs)的形成。AKR1C3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因子11P-PGF2。这在氧化应激和COPD之间提供了额外的潜在联系，并为新的治疗策略提供了可能性，其中包括AKR1C3抑制。最后，初步研究表明，只能由脂质过氧化产生的DNA加合物存在于吸烟者的尿液中，而不吸烟者的尿液中则完全没有。我们建议在这些令人兴奋的新发现的基础上，通过开发暴露和生物反应的体内生物标记物小组，我们假设这将使区分一组不吸烟的人和一组没有疾病的烟草吸烟者成为可能。目的1.探讨苯并[a]磷和苯并[a]P-7，8-二酮是否诱导NHBE细胞AKR1C/2，增加氧化应激形成DNA中的8-oxo-dGuo和HedGuo，诱导HASM细胞中的AKR1C3，增加PAH暴露的潜在尿液和EEC生物反应生物标志物--支气管收缩因子11p-PGF2的生物合成。目的：寻找苯并[a]P及其氧化代谢产物处理NHBE和HASM细胞后的分泌蛋白，作为PAH暴露的潜在血清生物反应生物标志物。目的：对尿液中的体内暴露和反应生物标志物以及外周血和血清中的生物反应标志物进行预测性和精细化分析，以区分非吸烟者和无疾病吸烟者。这项拟议研究的成功完成将提供一组暴露和对烟草烟雾的生物反应的生物标志物，将在未来旨在阐明基因环境相互作用与癌症、心血管疾病和COPD等疾病之间关系的研究中发挥重要作用。

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.