Mutagenicity/Genotoxicity with Endogenous PGH Synthase-2

内源性 PGH 合酶 2 的致突变性/基因毒性

• 批准号: 6405320
• 负责人: Hyesook Kim
• 金额: $ 37.62万
• 依托单位: DETROIT R & D, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6405320
• 关键词: DNA damage; DNA repair; benzopyrenes; cell line; colorimetry; fibroblasts; fluorescence; luminescence; mutagens; polymerase chain reaction; prostaglandin endoperoxide synthase; technology /technique development; transfection

DESCRIPTION (provided by applicant): Prostaglandin H synthase (PGHS) plays a significant role in inflammation and carcinogenesis. PGHS form 2 (PGHS-2) induced by mitogens, growth factors and tumor promotors causes mutagenicity and genotoxicity. A bacterial test is not suitable because reactive toxicants produced by PGHS-2 are short-lived and cannot easily cross the bacterial membrane. Our objective is to develop colorimetric/chemiluminescent and fluorescent real-time quantitative PCR (QPCR) assays to quantitate DNA damage mediated by endogenous human PGHS-2. In Phase I, we expressed human PGHS-2 in human DNA repair deficient fibroblast cell line, XPA, treated the cells with benzo(a)pyrene-7,8-dihydrodiol and measured DNA damage by QPCR of 16.2 kb mitochondrial DNA using colorimetric/chemiluminescent assays. In Phase II, we will improve the colorimetric/chemiluminescent QPCR assay internal control DNAs and develop a fluorescent real-time QPCR assay. We will also develop QPCR assays with a DNA damage repair-proficient human fibroblast cell line after transfection of the cells with a PGHS-2 gene-containing vector. QPCR assays will also be developed with human PGHS-1 expressing XPA. Utilities of the assays will be explored by treatment of the cells with several environmental toxicants. PROPOSED COMMERCIAL APPLICATION: Increased expression of PGHS-2 may lead to development of human tumors including colon cancer. Reliable mutagenicity and genotoxicity assays with PGHS-2 expressed in eukaryotic cells is in great demand. Thus, facile colorimetric/ECL and fluorescent Real-Time quantitative PCR (QPCR) assays will be developed with human PGHS-2 :expressed in human DNA repair deficient and proficient fibroblasts. The QPCR assay kits will be marketed to screen PGHS-2-dependent promutagens and DNA damaging agents as well as chemopreventive agents.

描述（由申请人提供）：前列腺素 H 合酶 (PGHS) 起着 在炎症和癌变中发挥重要作用。 PGHS 表 2 (PGHS-2) 由有丝分裂原、生长因子和肿瘤促进剂诱导，导致致突变性和 遗传毒性。细菌测试不适合，因为反应性毒物 PGHS-2 产生的细菌寿命较短，不易穿过细菌 膜。 我们的目标是开发比色/化学发光和荧光 实时定量 PCR (QPCR) 检测可定量 DNA 介导的损伤 内源性人PGHS-2。在第一阶段，我们在人类 DNA 中表达人类 PGHS-2 修复缺陷的成纤维细胞系 XPA，用 苯并(a)芘-7,8-二氢二醇并通过 16.2 kb 的 QPCR 测量 DNA 损伤 使用比色/化学发光分析法检测线粒体 DNA。 在第二阶段，我们将改进比色/化学发光 QPCR 检测 内部对照 DNA 并开发荧光实时 QPCR 测定。我们将 还开发了具有 DNA 损伤修复能力的人成纤维细胞的 QPCR 检测 用含有PGHS-2基因的载体转染细胞后的细胞系。 QPCR 检测也将使用表达 XPA 的人类 PGHS-1 进行开发。公用事业 将通过用几种方法处理细胞来探索测定的方法 环境毒物。 拟议的商业应用： PGHS-2表达增加可能导致包括结肠癌在内的人类肿瘤的发生。人们迫切需要对真核细胞中表达的 PGHS-2 进行可靠的致突变性和基因毒性测定。因此，将使用人类 PGHS-2 开发简便的比色/ECL 和荧光实时定量 PCR (QPCR) 测定：在人类 DNA 修复缺陷和熟练的成纤维细胞中表达。 QPCR 检测试剂盒将上市用于筛选 PGHS-2 依赖性促诱变剂和 DNA 损伤剂以及化学预防剂。