【作者】 沈文艳；

【导师】 余应年；

【作者基本信息】 浙江大学 ， 生理学， 2007， 博士

【摘要】 苯并(a)芘(BaP)属于多环芳烃类(PAHs)环境化学污染物，主要由煤、烟草和其它一些有机化合物高温加热或燃烧不完全时产生。苯并(a)芘在体内经微粒体酶代谢活化后生成终致癌物7,8-二羟-9,10-环氧苯并(a)芘(benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide，BPDE)，其中反式-BPDE是代谢产物中活性最强的，具有亲电性碳原子活性基团，能与组成核酸的碱基和组成蛋白质的氨基酸的亲核基团共价结合形成加合物，损伤生物大分子的结构和功能，从而引发核苷酸切除修复，DNA跨损伤复制，细胞周期“校正点”等信号通路的激活，以及基因表达的改变。BPDE是一个全致癌物，两阶段致癌实验表明它既是致癌剂又是促癌剂。其中，BPDE在癌变发生启动期的机制已经得到了广泛研究，主要是导致DNA突变形成：BPDE第10位上的C与DNA鸟嘌呤的N~2位形成加合物(+)-trans-anti-BPDE-N~2-dG，它们将阻断DNA复制，通过细胞的DNA合成机构的旁路合成途径，即跨损伤DNA合成(translesion DNA synthesis，TLS)，细胞可以避免这些未经修复的损伤对细胞的致死性影响，但在损伤碱基的对面常插入错配的碱基，通常是G→T的碱基颠换突变，导致细胞面临基因突变的风险。与BPDE的致突变机制广泛研究相反的是，BPDE诱发的细胞早期反应中是如何影响信号转导通路从而激活转录因子和它们的靶基因表达所知甚少。现在的研究发现BPDE作用后p53，PI-3K／Akt／JNKs，MAPKs／AP-1，IKKbeta／NF-kappaB等信号通路被激活。为了研究BPDE对基因表达的直接影响，用DNA免疫沉淀技术鉴定并克隆BPDE结合的DNA片段，总共得到67个基因片段。这些片段经测序并与GenBank数据库BLAST之后，发现其功能包括DNA修复，凋亡相关，锌指蛋白，酶，表达序列标签克隆，CpG岛。这些数据进一步说明BPDE与相关基因DNA直接结合也是诱导基因表达变化的一个重要机制。与此同时，高通量的基因芯片技术也被用于检测BPDE暴露后，细胞中基因发生的全局变化和涉及的信号通路。Akerman等用含有350个人类基因的芯片发现BPDE处理后改变的基因涉及：细胞周期调控，谷胱甘肽解毒，细胞凋亡等等。在毒理学研究中，我们通常要考虑细胞或动物暴露于环境污染物后的剂量反应关系。但是，用能诱导非毒性、亚毒性或毒性反应的不同浓度的外来污染物作用后，有时候不一定能得到良好的剂量依赖关系，甚至于能诱导机体产生完全不同的反应。Moller等人的研究结果发现用低、中、高三个浓度道诺红菌素(daunorubicin)处理细胞后，差异蛋白质组显示道诺红菌素能导致20个蛋白的表达水平在三个浓度都上调，大部分不具有剂量依赖关系。除了暴露剂量，暴露后的时间间隔也是一个需要考虑在内的重要因素。随着暴露后时间的延长，可以发现与时间相关毒性反应的可观察的基因或蛋白表达改变，有助于了解早期反应和晚期反应的作用机制和寻找相关的生物标记物。我们实验室曾用苯并[a]芘处理细胞，引起广泛的蛋白表达改变，其中以锌指蛋白和一些参与转录调控的蛋白多见。为了消除苯并[a]芘的代谢转化效率差异的影响，我们随后采用苯并[a]芘的代谢终产物BPDE进行深入研究，用低浓度0.005μM BPDE处理FL细胞，在细胞外液中发现有3个出现的蛋白点，16个表达上调的蛋白点。为了更好的了解细胞反应的全貌，实验中还用不同的暴露剂量以期获得更多的信息来阐明细胞对BPDE的应答机制。应用基因芯片技术分别检测0.005μM、0.05μM、0.5μMBPDE暴露后基因表达的改变并用实时定量RT-PCR进行验证，发现在低浓度0.005μM和0.05μM浓度组发生改变的基因较少，而高浓度0.5μM发生改变的基因数目较多，这些基因的功能涉及细胞周期调节、信号转导、转录因子、代谢有关的酶等。可能涉及的信号通路包括：p53，MAPK，Akt／PKB。刘更等发现用低浓度0.005μM和0.05μM BPDE可以诱导内质网应激反应(endoplasmic reticulum stress，ER stress)，但较高浓度0.5μM BPDE未见类似变化。以上这些实验结果使我们迫切想揭示不同浓度BPDE暴露和暴露后不同时间细胞发生的变化。近年来，生物技术的飞速发展使得更全面广泛的了解这种变化成为可能。许多高通量技术如cDNA芯片，实时RT—PCR，以及生物信息学已被用于细胞应激反应，实验结果令人鼓舞。但是这些技术都不能提供转录后的尤其是蛋白质在翻译后修饰的信息，而蛋白质通常是细胞内的功能执行者。以双向电泳(2-DE)作为分离技术和质谱作为鉴定技术的蛋白质组学的方法能同时分离细胞内几千个蛋白，并能研究翻译后修饰以及蛋白的相互作用，因此在相关领域中受到广泛的应用。因此，在本研究中，应用双向电泳和基质辅助激光解吸飞行时间质谱(MALDI-TOF)相结合的蛋白质组学方法来筛选不同浓度BPDE暴露和暴露后不同时间哺乳动物细胞的差异表达蛋白，研究可能存在的差异表达蛋白与BPDE浓度、作用时间的关系，找寻对BPDE有特异反应的蛋白。通过生物信息学方法对差异蛋白进行分类，探讨不同功能蛋白对BPDE的响应可能对细胞产生的毒性影响。我们用BPDE处理FL细胞，二甲基亚砜作为溶剂对照组，然后提取细胞总蛋白。采用24cm，pH 4-7的IPG胶条及同时可跑12块胶的Ettan DALTⅡ垂直电泳系统进行分离，银染的胶经数字化成像和图象分析后发现：量效关系中有65个蛋白斑点在BPDE处理后12小时发生了显著的变化。其中有1个斑点在0.05μM和0.5μM BPDE处理后的细胞中检测到，1个斑点仅在0.5μM BPDE处理后的细胞中检测到；另外有24个蛋白斑点在0.005μM BPDE处理后表达量发生改变(11个上调，13个下调)，24个蛋白斑点在0.05μM BPDE处理后表达量发生改变(16个上调，8个下调)，25个蛋白斑点在0.5μM BPDE处理后表达量发生改变(14个上调，11个下调)，有10个蛋白斑点在两个处理剂量组都发生了改变，没有蛋白斑点在三个剂量组都发生改变。进一步的，我们切取胶上的差异蛋白斑点，然后用特异性的胰酶消化，酶解后的肽段用基质辅助的激光解吸离子化-飞行时间质谱(MALDI-TOF)鉴定，得到各个蛋白的肽质指纹图谱(peptide mass fingerprinting，PMF)。根据肽质指纹图，在网上用MASCOT搜索软件分别搜寻NCBI的非冗余蛋白序列数据库(nrNCBI)，46个蛋白质得到了鉴定。这些得到成功鉴定的蛋白质功能涉及面非常广，包括转录调控，细胞周期，细胞增殖，信号转导，细胞骨架，发育，代谢以及其他功能未明的等等。表明BPDE对细胞产生了广泛的影响。在时效研究中发现用0.05μMBPDE处理后3小时，12小时和24小时，一共有128个蛋白斑点的表达发生了改变。36个蛋白斑点在0.05μM BPDE处理后3小时表达量发生改变(11个上调，25个下调)，29个蛋白斑点在0.05μM BPDE处理后12小时表达量发生改变(20个上调，9个下调)，69个蛋白斑点在0.05μMBPDE处理后24小时表达量发生改变(35个上调，34个下调)，有6个蛋白斑点在两个处理时间点都发生了改变，没有蛋白点在三个处理时间点都发生改变。其中84个斑点得到了成功鉴定。与量效研究相似的是，时效研究中这些得到成功鉴定的蛋白质功能涉及面也非常广，几乎涵盖了量效中改变蛋白的各个类别。结论：BPDE作用后细胞内发生了广泛的变化，有众多蛋白参与了应答反应，这些蛋白涉及到细胞的不同功能，为进一步研究BPDE与机体交互作用的分子机制提供了线索，并为寻找人群接触环境致癌物后的生物标记物提供可能。研究未发现蛋白有剂量依赖关系和时间依赖关系，说明不同浓度BPDE暴露及暴露后不同时间细胞应答反应机制是不一样的，高浓度BPDE作用后，细胞应答反应体系不是低浓度作用后的简单放大，存在更为复杂的致毒机制，不同浓度BPDE暴露及暴露后不同时间，可能激活细胞内不同的途径来产生毒性效应。更多还原

【Abstract】 Benzo(a)pyrene is one of the polycyclic aromatic hydrocarbons(PAHs) environmental pollutants and can be generated from incomplete combustion of organic materials such as gasoline in motor vehicles, coal burning, cooking, and tobacco smoke. Benzo(a)pyrene is metabolically activated to form the ultimate carcinogen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) by microsome enzymes. Anti-BPDE is the most carcinogenetic form and the electrophilic species of it is able to interact with nucleophilic sites on cellular DNA, RNA and protein resulting in bulky-adduct damage. As a result, nucleotide excision repair, translesion DNA synthesis, activation of cell cycle checkpoint pathway and alterations of gene expression are induced.BPDE is a complete carcinogen, which plays a role in tumor initiation and promotion in two-stage carcinogenesis test. Extensive studies have been done in the mechanism of BPDE in mutagenesis. DNA damage occurs mainly by binding of C 10 position of anti-BPDE with N~2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N~2-dG. The bulky adduct will block DNA replication. However, a DNA synthesis bypass way, translesion DNA synthesis can help cell to avoid the deadly fate of un-repaired DNA damage. As a result, mispairing base is often inserted opposite the lesion, which increases the risk of mutagenesis mainly G→T transversion mutation。However, the mechanism involved in the early responses induced by BPDE, which is thought to be mediated through initiating signal transduction pathways leading to activation of transcription factors and their target genes, is barelyunderstood.Studies have shown signaling pathway such as p53, PI-3K/Akt/JNKs, MAPKs/ AP-1, IKKbeta/NF-kappaB was activated after BPDE exposure. To investigate the direct effects of BPDE on gene expression, DNA immunoprecipitation technique was used to identify and clone BPDE-binding DNA fragments. A total of 67 fragments were sequenced and BLASTed in the GenBank database. The 67 fragments include DNA repair and apoptosis-related genes, zinc finger protein, cellular enzymes, expressed sequence tag clones, and CpG islands. These data further demonstrate that direct binding of BPDE with DNA of related genes is an important mechanism of BPDE-induced alterations in gene expression。 In addition, high-throughput gene microarray has been applied to detect the cellular global changes in gene and signaling pathways. After exposure to BPDE, Akennan et al used a human 350 gene array and found the genes with alterated expression were involved in cell cycle regulation, glutathione detoxification, cell apoptosis and etc.In study of responses to toxins, dose-related responses should be well considered. However, different doses belong to nontoxic, subtoxic or toxic ranges may actually induce dose dependent or completely different responses. In a proteomics study, Moller et al. found that treatment with daunorubicin of low, medium and high dose led to a significant up-regulation of 20 proteins, independent of the concentration used.In addition to exposure dose, the intervals after exposure is also should be taken into accounted. While the intervals elongates, the time related alteration in gene or protein expression could be discovered. It provides the possibility for the understanding of the mechanisms and finding of the biomarkers of the early and late response.In our laboratory, we have performed proteomic analysis in B[a]P-treatment in human amnion epithelial cells and a comprehensive alteration of protein expression profiles were found. The identified proteins included a number of zinc finger proteins and other transcription regulators. In order to eliminate the potential confounding effects of differential metabolic activation, we used the reactive metabolites of B[a]P for further study. Human amnion cells (FL cells) were exposed to 0.005μM BPDE in a secretome proteomic experiment, as a result, 3 and 16 protein spots were foundappearing and up-regulated in the cultured medium of BPDE treated cells as compared to the control group. Oligonucleotide microarray technique was also applied to detect the differential gene expression profiles after exposure to 0.005μM, 0.05 and 0.5μM BPDE, followed by quantitative real-time RT-PCR validation. There were few and robust gene expression changes, respectively, in response to the two lower doses and the higher dose of BPDE. The results in high dose exposure showed the alteration in expression level of genes related to cell cycle control, signaling molecules, transcription factors, and metabolic enzymes etc., signaling pathways such as p53-mediated, mitogen-activated protein kinases, MAPKs, Akt/PKB were activated. Additionally, Liu et al found 0.005μM and 0.05μM BPDE could trigger the endoplasmic reticulum stress in exposed cells, while 0.5μM BPDE could not induce such a response.All these findings raised our interests in revealing the cellular changes at different concentrations and time intervals after exposure. The rapid development of biological techniques has given us the possibility to try to understand the changes more comprehensively. Nowadays, many high-throughput methods including gene expression microarray assay, real-time RT-PCR analysis and bioinformatics are under way in this laboratory for the study of cell stress responses, and the preliminary results are inspiring. But these methods cannot provide direct information of how proteins are regulated at the translational or post-translational levels. Since proteins rather than mRNA are usually the functional executors in cells. Proteomic analysis, which combines 2-DE and mass Spectrometry, allows simultaneous monitoring of the expression of hundreds and even thousands of proteins in a sample following exposure to toxicant. Furthermore, it can study the post translation modification and protein interaction. Therefore, proteomic analysis is becoming a popular high throughput method of choice to detect differentially expressed proteins between profiles after exposure to toxicants.In our present studies, 2-DE combining MS was undertaken to identify the differentially expressed proteins following exposure to different doses of BPDE as well as at different time intervals after BPDE exposure. The differentially expressed proteins were classified according to their function through bioinformatics analysisand the functional implications of these proteins were discussed.In order to identify the proteins of differential expression in the FL cells after BPDE treatment, the IPG strips (24 cm, pH 4-7) and Ettan DALT II vertical electrophoresis system which allow a higher loading capacity and provide a longer separation distance for micropreparative runs were used to separate the whole cellular proteins of the FL cells treated with BPDE and DMSO, respectively. Then the 2D gels were visualized by silver staining; the digitized images were analyzed with 2D analysis software. In dose response study, 65 protein spots showed significant changes in BPDE-treated cells compared to control cells (DMSO treatment). There was 1 protein spot detected after 0.05μM and 0.5μM BPDE treatment, 1 protein spot detected only after 0.5μM BPDE treatment. Moreover, another 24 protein spots were found to be affected by 0.005μM BPDE(11 were up-regulated, 13 were down-regulated), 24 protein spots were found to be affected by 0.05μM BPDE(16 were up-regulated, 8 were down-regulated), 25 protein spots were found to be affected by 0.5μM BPDE(14 were up-regulated, 11 were down-regulated); however,10 protein spots were found to be affected by BPDE in two doses, no protein spot was affected by BPDE in three doses. These protein spots were cut from the gels and subjected to in-gel digestion with trypsin. The peptides were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass Spectrometry to obtain the peptide mass fingerprinting (PMF). And based on the information of PMF, some of the proteins were identified by searching non-redundant NCBI protein database using MASCOT software. Forty six protein spots were identified successfully. These identified proteins are involved in a variety of cellular process including transcription regulation, cell-cycle control, cell proliferation, signal transduction, cell skeleton, development, metabolism and some proteins with unknown functions. Based on the result, BPDE exposure induced comprehensive cellular responses.One hundred and twenty eight protein spots showed significant differential expression in the time response study. 36 protein spots were found to be affected by 0.05μM BPDE(11 were up-regulated, 25 were down-regulated) at 3 hour after exposure, 29 protein spots were found to be affected by 0.05μM BPDE(20 wereup-regulated, 9 were down-regulated) at 12 hour, 69 protein spots were found to be affected by 0.05μM BPDE(35 were up-regulated, 34 were down-regulated)at 24 hour; however,6 protein spots were found to be affected by BPDE in two time intervals, no protein spot was affected by BPDE in three time intervals. Eighty four protein spots were identified successfully. Similar to the dose response study, these identified proteins are involved in a variety of cellular process.Conclusion: There are comprehensive responses in the FL cells after exposure to BPDE. Many proteins are involved in BPDE-induced cellular responses in mammalian cells. These proteins take part in a variety of cellular processes. This work provides the new insight into the mechanisms of BPDE and the possibility of new biomarkers for evaluating the exposure to environmental carcinogen. Little dose-dependent or time-dependent manner could be observed in the proteins in the dose response study or time response study. Therefore, the biological responses of high dose exposure can’t be considered as only an amplification of low dose response. BPDE may activate different pathway to regulate the cell response machinery followed exposure to different doses as well as at different time intervals after exposure.更多还原