Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

Abstract

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine

“…Benzylic alcohols derived from polycyclic aromatic hydrocarbons have been evaluated extensively by these recombinant sulfotransferase bacteria and V79 cells [Czich et al, 1994;Glatt et al, 1998]. One line, V79-MZ-rCYP1A2, expressing both rat cytochrome P4501A2 [Wölfel et al, 1991] and a male-specific rat sul- Open and closed symbols of the same shape represent separate experiments. The UV5P3 data in both survival and mutations experiments were replotted from a previous study .…”

“…Benzylic alcohols derived from polycyclic aromatic hydrocarbons have been evaluated extensively by these recombinant sulfotransferase bacteria and V79 cells [Czich et al, 1994;Glatt et al, 1998]. One line, V79-MZ-rCYP1A2, expressing both rat cytochrome P4501A2 [Wo¨lfel et al, 1991] and a malespecific rat sul-fotransferase [Nagata et al, 1993], was used to evaluate the cytotoxicity and mutagenicity of 2acetylaminofluorene at the hprt locus [Glatt et al, 1998]. The present study is the first to stably express the HAST1 and HAST3 sulfotransferases in the repair deficient CHO cell line.…”

Predicting Drug Metabolism–dependent Toxicity for Humans with a Genetically Engineered Cell Battery

“…In 1986, bearing all this in mind, we started to generate an in vitro tool based on genetically engineered, established cells for the stable and indi- By 1987, we had the first cell line running and expressing the rat CYP2B1 in a functional state (1), followed by various other CYPs from rat over the next five years (5,6). Based on this success, in 1991, we had the courage to approach the cloning of human CYPs, eventually ending up with all the human CYPs relevant for drugs and chemicals (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”