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| Grant Number: | 5R44ES011251-03 |
| PI Name: | KIM, HYESOOK S. |
| PI Email: | hskim@detroitrandd.com |
| PI Title: | |
| Project Title: | Mutagenicity/Genotoxicity with Endogenous PGH Synthase-2 |
Abstract: 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.
Thesaurus Terms:
DNA damage, colorimetry, luminescence, mutagen, polymerase chain reaction, prostaglandin endoperoxide synthase, technology /technique development
DNA repair, benzopyrene, fluorescence
cell line, fibroblast, transfection
| Institution: | DETROIT RESEARCH AND DEVELOPMENT, INC. |
| 4683 RAVINE DR | |
| BLOOMFIELD HILLS, MI 48301 | |
| Fiscal Year: | 2002 |
| Department: | |
| Project Start: | 30-SEP-2001 |
| Project End: | 30-JUN-2003 |
| ICD: | NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES |
| IRG: | ZRG1 |