Transfection of a Rat Cytochrome P450b cDNA Into C3H10T1/2CL8 Mouse Embryo Fibroblasts

Abstract

A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. This bifunctional plasmid (pJRSL100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418-resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2-acetylaminofluorene (2-AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b-4, exhibited an increased cytotoxic response to 2-AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.

A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity testing

“…But structural information about the types of complexes formed by meso-DiMeDMSA and Cd2+ ions, and direct chemical evidence that meso-DiMeDMSA will coordinate Cd2+ ions in the presence of metallothionein, has been lacking. Cadmium and thiolato ligands can form complexes that are either mononuclear (8,9) or polynuclear (10)(11)(12)(13)(14). The formation of polynuclear complexes has stimulated a large amount of research in the area of metal-thiolate chemistry.…”