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

• 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
• 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

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.

固体纳米孔和深度测序(PQ3)测定BPDE-DNA加合物 强致癌物质苯并[a]芘(BaP)存在于严重污染的空气、烟熏食品和烟草烟雾中。一旦进入细胞，BaP可以形成稳定的苯并[a]芘二氢二醇环氧化物(BPDE)DNA加合物，导致在复制过程中插入错误的碱基。BPDE-DNA加合物被认为是暴露于BaP的生物标志物，白细胞以及肺和食道等组织中BPDE-DNA加合物浓度的增加与癌症风险增加有关。目前测量BPDE加合物的方法要么使用高放射性试剂且非特异性，要么需要高度专业化的设备和令人望而却步的大量DNA来进行大多数流行病学研究。用于基因组BPDE加合物的特定生物监测的新型传感器不需要危险的试剂，也不需要繁琐的程序，将加快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座位上的加合物与各种癌症的关联。BPDE与基因组DNA的结合不是随机的。最近的报道表明，BPDE-DNA复合体优先分布在甲基化的CpG二核苷酸上，以及抑癌基因p53和ras癌基因的突变热点上。然而，用现有的方法，BPDE加合物只能在非常有限的基因组座位上被检测。这样的调查将需要使用高放射性试剂和测序凝胶的繁琐技术。用于估计基因组上BPDE浓度的新的非密集方法可以提供新的工具，用于确定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。这项提议的目标是开发新的固态传感器和新的“OMIC”式的DNA加合物测量方法。这两种方法都将专门针对BPDE-DNA测量进行调整，并将具有使用外科组织进行后续人类癌症研究所需的灵敏度，而不需要令人望而却步的大量DNA。目标1将开发一种用于BPDE-DNA加合物单分子检测的固态纳米孔测量系统。AIM 2将开发一种方案，用于绘制暴露于BaP或BPDE的细胞株DNA中BPDE加合物的基因组图谱。这项研究将提供“概念证明”和科学证据，在此基础上可以设计后续实验，以评估特定人群中的苯并苯暴露。实现这些目标将开辟新的研究领域，并为快速和详细地测量长期和短期暴露于苯并苯的影响提供有价值的新工具。这里介绍的方法可以扩展到形成DNA加合物的大多数其他毒素和致癌物质。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。