Mutagenicity/Genotoxicity with Endogenous PGH Synthase-2

描述（由申请人提供）：前列腺素H合酶（PGHS）起着重要的作用。 在炎症和致癌中起重要作用。PGHS表格2（PGHS-2） 由有丝分裂原、生长因子和肿瘤促进剂引起的致突变性， 遗传毒性细菌试验不适用，因为反应性毒物 PGHS-2产生的细菌毒素是短暂的，不能轻易地穿过细菌。 膜的 我们的目标是开发比色/荧光和荧光 实时定量PCR（QPCR）测定，以定量由 内源性人PGHS-2。在第一阶段，我们在人DNA中表达人PGHS-2， 修复缺陷型成纤维细胞系XPA用以下物质处理细胞 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/荧光测定法测定线粒体DNA。 在第二阶段，我们将改进比色/荧光QPCR检测 内部对照DNA并开发荧光实时QPCR测定。我们将 我还开发了QPCR检测与DNA损伤修复熟练的人成纤维细胞 用含有PGHS-2基因的载体转染细胞后的细胞系。 还将使用表达XPA的人PGHS-1开发QPCR试验。公用事业 将通过用几种不同的细胞处理细胞来探索这些测定法。 环境毒物 拟定商业应用： PGHS-2的表达增加可能导致包括结肠癌在内的人类肿瘤的发展。在真核细胞中表达的PGHS-2的可靠的致突变性和遗传毒性试验是非常需要的。因此，将使用人PGHS-2开发简便的比色/ECL和荧光实时定量PCR（QPCR）试验：在人DNA修复缺陷型和熟练型成纤维细胞中表达。QPCR检测试剂盒将上市，用于筛选PGHS-2依赖性前诱变剂和DNA损伤剂以及化学预防剂。

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.