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.
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.
