Measurements of BPDE-DNA adducts by solid state nonopore & deep sequencing (PQ3

通过固态非孔测量 BPDE-DNA 加合物

基本信息

批准号：
8384743
负责人：
Rashid Bashir
金额：
$ 18.01万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-20 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8384743
关键词：
Air Area Benzene Benzo(a)pyrene Binding Bioinformatics Biological Biological Markers Biological Monitoring Caliber Cancer Etiology Carcinogens Cell Line Cells Complex CpG dinucleotide DNA DNA Adducts DNA Footprint Detection Development Engineering Epidemiologic Studies Epigenetic Process Epoxy Compounds Equipment Esophageal Exposure to Food Gel Genome Genome Mappings Genomics Goals Guanine Nucleotides Health Human International Agency for Research on Cancer Investigation Knowledge Leukocytes Link Lung Malignant Neoplasms Maps Measurement Measures Methodology Methods Molecular Biology Mutation Operative Surgical Procedures Outcomes Research Population Principal Investigator Procedures Protein p53 Protocols documentation Radioactive Reaction Reagent Reporting Research Resolution Risk Role Sampling Scientist Smoke Speed System Techniques Testing Tissues Tobacco smoke Toxicology Toxin adduct base cancer initiation cancer risk design improved multidisciplinary nanopore nanoscale novel programs ras Oncogene research study sensor single molecule solid state tool

项目摘要

Measurements of BPDE-DNA adducts by solid state nanopore and deep sequencing (PQ3) Potent carcinogen Benzo[a]pyrene (BaP) is found in heavily polluted air, smoked food, and tobacco smoke. Once inside cells, BaP can form stable benzo[a]pyrene dihydrodiol epoxide (BPDE) DNA adducts leading to the insertion of incorrect bases during replication. BPDE-DNA adducts are considered biomarkers of exposure to BaP and increased concentrations of BPDE-DNA adducts in white blood cells and in tissues such as lung and esophageal are associated with increased risk of cancer. Current methods for BPDE adduct measurements either involve highly radioactive reagents and are non-specific, or require highly specialized equipment and prohibitively large amounts of DNA for most epidemiological studies. Novel sensors for specific biomonitoring of genomic BPDE adduct which do not require hazardous reagents, or cumbersome procedures will speed the links between BaP exposure and development of various cancers. Furthermore, it is difficult to assess the associations of adduct at specific genomic loci and various cancers. The binding of BPDE on the genomic DNA is not random. Recent reports have demonstrated a preferential distribution of the BPDE-DNA complexes on the methylated CpG dinucleotides and on the mutational hotspots in tumor suppressor p53 and ras oncogene. However, with the existing methodologies BPDE adducts can only be examined at a very limited number of genomic loci. Such investigations would require laborious techniques involving highly radioactive reagents and sequencing gels. Novel non-intensive methodologies for estimating BPDE concentrations on the genome could provide new tools for identifying potential links between BaP exposure and specific mutations and epigenetic alterations that cause cancer. The objective of this proposal is to develop new solid state sensors and novel "omic" -style methodologies for the measurement of DNA adducts. Both approaches will be specifically tuned to BPDE-DNA measurements and will have the sensitivity needed for subsequent studies in human cancer using surgical tissues without requiring prohibitively large amounts of DNA. Aim 1 will develop a solid state nanopore measurement system for single molecule detection of BPDE-DNA adducts. Aim 2 will develop a protocol for genome mapping BPDE adducts in DNA from cell lines exposed to BaP or BPDE. This research will provide the "proof of concept" and scientific evidence upon which subsequent experiments can be designed for assessing BaP exposures in specific populations. Achieving these goals will open new areas of research and provide valuable new tools for fast and detailed measurements of long and short term exposure to the BaP. The approaches introduced here can be extended to most other toxins and carcinogens that form DNA adducts. These findings will benefit diverse areas of cancer and scientific research, such as toxicology, where adducts are studied.

通过固态纳米孔和深度测序测量 BPDE-DNA 加合物 (PQ3) 在严重污染的空气、熏制食品和烟草烟雾中发现了强效致癌物质苯并[a]芘 (BaP)。一旦进入细胞内，BaP 就可以形成稳定的苯并[a]芘二氢二醇环氧化物 (BPDE) DNA 加合物，导致复制过程中插入错误的碱基。 BPDE-DNA 加合物被认为是接触 BaP 的生物标志物，白细胞以及肺和食管等组织中 BPDE-DNA 加合物浓度的增加与癌症风险增加相关。目前的 BPDE 加合物测量方法要么涉及高放射性试剂且非特异性，要么需要高度专业的设备和对于大多数流行病学研究而言令人望而却步的大量 DNA。用于基因组 BPDE 加合物特异性生物监测的新型传感器不需要危险试剂或繁琐的程序，将加速 BaP 暴露与各种癌症发展之间的联系。此外，很难评估特定基因组位点处的加合物与各种癌症的关联。 BPDE 在基因组 DNA 上的结合不是随机的。最近的报告表明，BPDE-DNA 复合物优先分布在甲基化 CpG 二核苷酸以及抑癌基因 p53 和 ras 癌基因的突变热点上。然而，使用现有的方法只能在非常有限的基因组位点上检查 BPDE 加合物。此类研究需要费力的技术，涉及高放射性试剂和测序凝胶。用于估计基因组上 BPDE 浓度的新型非密集方法可以提供新工具，用于识别 BaP 暴露与导致癌症的特定突变和表观遗传改变之间的潜在联系。该提案的目标是开发新的固态传感器和新颖的“组学”式方法来测量 DNA 加合物。这两种方法都将专门针对 BPDE-DNA 测量进行调整，并且具有使用手术组织进行人类癌症后续研究所需的灵敏度，而不需要大量的 DNA。目标 1 将开发一种固态纳米孔测量系统，用于 BPDE-DNA 加合物的单分子检测。目标 2 将开发一种方案，用于对暴露于 BaP 或 BPDE 的细胞系 DNA 中的 BPDE 加合物进行基因组定位。这项研究将提供“概念证明”和科学证据，随后可以设计实验来评估特定人群的 BaP 暴露情况。实现这些目标将开辟新的研究领域，并为快速、详细地测量 BaP 的长期和短期暴露提供有价值的新工具。这里介绍的方法可以扩展到大多数其他形成 DNA 加合物的毒素和致癌物。这些发现将有利于癌症和科学研究的各个领域，例如研究加合物的毒理学。