Defining PAH-mediated carcinogenesis in lung: CYP metabolism and B[a]P transport

定义 PAH 介导的肺癌致癌作用：CYP 代谢和 B[a]P 转运

基本信息

批准号：
7502209
负责人：
Stacy Lynn Gelhaus
金额：
$ 4.96万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-30 至 2010-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7502209
关键词：
2&apos -deoxyadenosine ABCB1 gene ABCC1 gene Address Adipose tissue Amines Amino Acids Animals Area Aromatic Polycyclic Hydrocarbons Benz(a)Anthracenes Benzo(a)pyrene Binding Biological Biological Assay CYP1A1 gene CYP3A4 gene CYP3A5 gene Cancer Etiology Carcinogens Cations Cattle Cause of Death Cell Line Cell Nucleus Cells Characteristics Coal CpG Islands Cultured Cells Cytochrome P450 Cytochromes DNA DNA Adduction DNA Adducts DNA Damage Data Deoxyguanosine Development Dimethyl Sulfoxide Dioxins Drug Delivery Systems Drug Metabolic Detoxication Environmental Carcinogens Environmental Pollutants Enzymes Epoxide hydrolase Experimental Models Exposure to Family member Fire - disasters Food Garbage Gases Genomics Glutathione Glutathione S-Transferase Glycol Glycoproteins Guanine Hepatocyte Human Human Milk Hydrolysis In Vitro Individual Individual Differences International Agency for Research on Cancer Itraconazole Kidney Link Liquid Chromatography Literature Liver Lung Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of urinary bladder Mass Spectrum Analysis Meat Mediating Messenger RNA Metabolic Metabolic Activation Metabolic Pathway Metabolism Methodology Methods Modeling Monitor Multidrug Resistance Associated Protein 1 Mus Mutation Names Nature Null Lymphocytes Oils Oxides P-Glycoprotein Pathway interactions Pharmaceutical Preparations Phase Play Point Mutation Promoter Regions Protein Isoforms Protein p53 Proteomics Public Health Pyrenes RNA Interference Reaction Reporting Research Reverse Transcriptase Polymerase Chain Reaction Role Skin Small Interfering RNA Smoke Smoker Stable Isotope Labeling TP53 gene Testing Tobacco Tobacco smoke Toxic effect Transfection Transferase United States United States Dept. of Health and Human Services Western Blotting Wood material Workplace adduct anticancer research base benzanthracene carbonyl reductase (NADPH)carcinogenesis cigarette smoking cigarette smoking cytochrome P450 3A extracellular fluoranthene hepatoma cell inhibitor/antagonist insight lung carcinogenesis multidrug resistance-associated protein 2 novel novel therapeutics prevent protein expression pyrene research study stable isotope tobacco exposure transcription factor

项目摘要

DESCRIPTION (provided by applicant): In this proposed research, benzo[a]pyrene (B[a]P) and it's metabolites, will be used as model compounds to determine the major metabolic pathways of PAHs in lung carcinogenesis. It is generally accepted that cytochrome P4501A1, CYP1A1, is primarily responsible for the metabolism of B[a]P to 7,8-dihydroxy-9,10- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate DNA-adduct forming, carcinogenic reactive metabolite; thus connecting CYP1A1 to the toxicological effects of this environmental carcinogen. DNA-adduct formation has been detected at CpG islands located in the promoter region of the tumor suppressor p53. Inactivation of p53 by point mutations is seen in over 40% of all cancers. However, in preliminary research using H358, human bronchoalveolar lung cells, it was found that DNA-adduct levels were higher in non-treated CYP1A1/1B1 null cells compared to H358 cells in which CYP1A1 expression was induced with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). This data correlates with literature results of CYP1A1 KO mice stuides in which it was determined that CYP1A1 conferred a protective rather than a toxic effect. This is indicative of the metabolic activity of another CYP in the metabolism of B[a]P. Several other CYP enzymes are capable of catalyzing this metabolism. Preliminary data has also indicated that TCDD may have an effect on the transport of B[a]P-reactive metabolites. It is known that TCDD is able to induce several of the ABCC drug transporter family members; therefore, explaining why less DNA-adduct formation is observed in lung cells pretreated with TCDD . This proposal, which has two aims, will identify the predominate CYP responsible for B[a]P metabolism in the lung and determine the role of B[a]PDE transporters in DNA-adduct formation in lung cells. A stable isotope dilution LC-MRM/MS method, previously developed, will be used for DNA-adduct quantification. DNA-adduct levels will be correlated to mRNA and protein expression determined by RT-PCR and a novel quantitative proteomics method, SI LAC. CYPs including 1A1, 1B1, 1A2, 2B6, 2E1, 2J2, 3A4, and 3A5 will be investigated. DNA-adduct formation will also be monitored in lung cells in which drug transporter expression has been inhibited by siRNA. Continuing research in this area will provide exciting new therapeutic strategies (drug targets) that can be employed to prevent PAH-mediated DNA damage. Relevance to public health: Cigarette smoke is known to cause 90% of lung cancer, but only 10% of smokers develop lung cancer. Examination of alternative CYP metabolism and B[a]PDE transport in lung cells will provide an experimental model to explore whether inter-individual differences in CYP metabolism and transport contribute to these phenomenon.

描述（由申请人提供）：在这项拟议的研究中，苯并[a]芘（B[a]P）及其代谢物将用作模型化合物，以确定多环芳烃在肺癌发生中的主要代谢途径。人们普遍认为细胞色素 P4501A1（CYP1A1）主要负责 B[a]P 代谢为 7,8-二羟基-9,10-环氧-7,8,9,10-四氢苯并[a]芘（B [a]PDE)，最终形成 DNA 加合物的致癌反应性代谢物；从而将 CYP1A1 与这种环境致癌物的毒理学作用联系起来。位于肿瘤抑制因子 p53 启动子区域的 CpG 岛已检测到 DNA 加合物形成。超过 40% 的癌症均因点突变而导致 p53 失活。然而，在使用人支气管肺泡肺细胞 H358 的初步研究中，发现与用 2、3、7、8 诱导 CYP1A1 表达的 H358 细胞相比，未处理的 CYP1A1/1B1 无效细胞中的 DNA 加合物水平较高。 -四氯二苯并-对-二恶英（TCDD）。该数据与 CYP1A1 KO 小鼠研究的文献结果相关，其中确定 CYP1A1 具有保护作用而不是毒性作用。这表明 B[a]P 代谢中另一种 CYP 的代谢活性。其他几种 CYP 酶能够催化这种代谢。初步数据还表明，TCDD 可能对 B[a]P 反应性代谢物的转运有影响。众所周知，TCDD 能够诱导多种 ABCC 药物转运蛋白家族成员；因此，解释了为什么在用 TCDD 预处理的肺细胞中观察到较少的 DNA 加合物形成。该提案有两个目标，将确定负责肺中 B[a]P 代谢的主要 CYP，并确定 B[a]PDE 转运蛋白在肺细胞 DNA 加合物形成中的作用。先前开发的稳定同位素稀释 LC-MRM/MS 方法将用于 DNA 加合物定量。 DNA 加合物水平将与 RT-PCR 和新型定量蛋白质组学方法 SI LAC 测定的 mRNA 和蛋白质表达相关联。将研究包括 1A1、1B1、1A2、2B6、2E1、2J2、3A4 和 3A5 在内的 CYP。还将监测肺部细胞中 DNA 加合物的形成，其中药物转运蛋白的表达已被 siRNA 抑制。该领域的持续研究将提供令人兴奋的新治疗策略（药物靶点），可用于预防 PAH 介导的 DNA 损伤。与公共健康的相关性：已知 90% 的肺癌是由香烟烟雾引起的，但只有 10% 的吸烟者会患上肺癌。对肺细胞中替代 CYP 代谢和 B[a]PDE 转运的检查将提供一个实验模型，以探讨 CYP 代谢和转运的个体间差异是否导致这些现象。