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Epigenetic toxicity of polycyclic aromatic hydrocarbons

多环芳烃的表观遗传毒性

基本信息

批准号：
7417349
负责人：
Brad L. Upham
金额：
$ 0.54万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-16 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7417349
关键词：
Affect Apoptosis Aromatic Hydrocarbons Aromatic Polycyclic Hydrocarbons Arts Atherosclerosis Biological Markers Carcinogens Cell Line Cell Proliferation Cell membrane Cells Chromatography Cigarette Class Classification Connexin 43 Data Development Diet Disease Dominant-Negative Mutation End Point Environment Environmental Tobacco Smoke Enzyme Inhibitor Drugs Enzyme Inhibitor Gene Enzyme Inhibitors Epigenetic Process Epithelial Epithelial Cells Event Excision Gene Expression Gene Silencing Glycyrrhetinic Acid Growth Growth and Development function High Pressure Liquid Chromatography Intercellular Communication Induction Isomerism Link Lipids Maintenance Malignant Neoplasms Marijuana Smoking Membrane Membrane Lipids Mitogen Activated Protein Kinase 1 Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Molecular Molecular Weight Pathway interactions Phospholipase Play Polycyclic Hydrocarbons Property Proteins Proteomics Proto-Oncogene Proteins c-akt RNA Interference Regulation Research Research Personnel Role Second Messenger Systems Series Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Molecule Signaling Protein Small Interfering RNA Stem cells Stimulation of Cell Proliferation Structure-Activity Relationship Techniques Technology Telomerase Testing Therapeutic Tobacco Toxic effect Toxicology Tumor Promotion U-0126 base cell growth cigarette smoking design human disease inhibitor/antagonist intercellular communication light scattering phospholipase inhibitor programs research study response second messenger

项目摘要

DESCRIPTION (provided by applicant): Cigarette smoking is an important cause of cancers of the lung, larynx, pharynx, esophagus, bladder, kidney, pancreas and more recently determined, the liver. Considerable toxicological research on polycyclic aromatic hydrocarbons (PAHs), which are prevalent compounds in cigarette smoke that contribute to cancer, has focused on their genotoxic attributes. However, many human diseases, such as cancer, are not solely the consequence of non-reversible mutagenic events but also include reversible, epigenetic events (altered expression of genes at transcriptional, translational and post-translational levels). Thus, there is a need to reassess the toxicity of PAHs at the epigenetic level. Gap junctional intercellular communication (GJIC) plays a central role in modulating signal transduction pathways that epigenetically alter gene expression. There is considerable evidence linking abnormal regulation of GJIC with the nongenotoxic steps of tumor promotion. Mitogen activated protein kinases (MAPKs) are also known to play a central role in cell signaling. We will use a series of tobacco smoke-relevant PAHs and determine structure activity relationships with inter- and intracellular signaling mechanisms in pluripotent mammalian, including human, epithelial cell lines. We will specifically test the hypothesis SA#1 that phospholipases are the upstream regulators of GJIC and MAPK in response to cigarette smoke-relevant PAHs by using specific phospholipase inhibitors and the emerging and powerful technique of silencing genes using small interfering RNA (siRNA). By using chromatographic techniques, we will test the hypothesis SA#2 that the phospholipases will release lipid-derived second messengers from the plasma membrane, and also the hypothesis that these messengers will activate signal transduction proteins that regulate GJIC and MAPK activity by using state of the art proteomic techniques to identify these unknown signaling proteins. We will also test the hypothesis SA#3 that alteration of GJIC will contribute to the mitogenic process and inhibit apoptosis and cell differentiation by monitoring genes and proteins specific to these biological endpoints using flow cytometry, and Western & Northern blot analyses in normal cell lines as well as those transfected with dominant negative gap junction genes or gap junction-siRNA. Our final hypothesis SA#4 to be tested will be that the effects of these PAHs on GJIC and MAPK will be the same on epithelial stem cell types from different tissues and species, which all express the same major gap junction protein. We will use our well-established F344-WB rat liver epithelial stem cell line, our newly isolated human liver stem cell line, a mouse lung epithelial cell line and a human bronchial epithelial cell line. We would like to note that the use of the biologically active 1-methyl isomer vs the inactive 2-methylisomer of anthracene, a tobacco smoke PAH, for all of the aims allows us to systematically identify molecular events that are specific to the regulation of GJIC and MAPK and subtract out non-specific events. Overall, determining the effect of tobacco-relevant PAHs on key signaling and gene expression events would provide invaluable mechanistically based information on the epigenetic toxicity of these compounds, thereby aiding in the development of preventative and therapeutic strategies of controlling human diseases such as cancer.

描述（由申请人提供）：吸烟是肺癌、喉癌、咽癌、食道癌、膀胱癌、肾癌、胰腺癌和最近确定的肝癌的重要原因。多环芳烃（PAHs）是香烟烟雾中常见的致癌化合物，大量的毒理学研究集中在其遗传毒性属性上。然而，许多人类疾病，如癌症，不仅是不可逆的诱变事件的结果，而且还包括可逆的表观遗传事件（在转录、翻译和翻译后水平上基因表达的改变）。因此，有必要在表观遗传水平上重新评估多环芳烃的毒性。细胞间隙连接通讯（GJIC）在调节表观遗传学改变基因表达的信号转导途径中起着重要作用。有相当多的证据表明GJIC的异常调节与肿瘤促进的非遗传毒性步骤有关。促分裂原活化蛋白激酶（MAPK）也已知在细胞信号传导中起中心作用。我们将使用一系列的烟草烟雾相关的多环芳烃，并确定结构活性关系与多能哺乳动物，包括人类，上皮细胞系间和细胞内的信号传导机制。我们将专门测试假设SA#1，即磷脂酶是GJIC和MAPK的上游调节剂，以响应香烟烟雾相关的多环芳烃，通过使用特定的磷脂酶抑制剂和新兴的和强大的技术沉默基因，使用小干扰RNA（siRNA）。通过使用色谱技术，我们将测试假设SA#2，即磷脂酶将从质膜释放脂质衍生的第二信使，以及这些信使将激活调节GJIC和MAPK活性的信号转导蛋白的假设，通过使用最先进的蛋白质组学技术来鉴定这些未知的信号蛋白。我们还将测试假设SA#3，即GJIC的改变将有助于促有丝分裂过程，并通过使用流式细胞术和Western &北方印迹分析在正常细胞系以及用显性负性间隙连接基因或间隙连接-siRNA转染的细胞系中监测对这些生物学终点特异的基因和蛋白质来抑制细胞凋亡和细胞分化。我们的最终假设SA#4将被测试，这些多环芳烃对GJIC和MAPK的影响将是相同的上皮干细胞类型从不同的组织和物种，都表达相同的主要间隙连接蛋白。我们将使用我们成熟的F344-WB大鼠肝上皮干细胞系、我们新分离的人肝干细胞系、小鼠肺上皮细胞系和人支气管上皮细胞系。我们想指出的是，使用蒽的生物活性1-甲基异构体与非活性2-甲基异构体，烟草烟雾PAH，为所有的目标，使我们能够系统地确定特定于GJIC和MAPK的调节的分子事件，并减去非特异性事件。总的来说，确定烟草相关的多环芳烃对关键信号和基因表达事件的影响将提供关于这些化合物的表观遗传毒性的基于机制的宝贵信息，从而有助于开发控制人类疾病如癌症的预防和治疗策略。