Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing.
V79 Chinese hamster cells genetically engineered to express cytochrome P-450IA1 are reported. A full length cDNA encoding rat cytochrome P-450IA1 was obtained from a cDNA library prepared from rat liver mRNA. The cDNA was recombined with the SV40 early promoter and expressed in V79 cells. Three V79-derived P-450IA1-expressing cell lines (XEM1, XEM2, and XEM3) were established. The presence of the rat cytochrome P-450IA1 cDNA in these hamster cells was confirmed by Southern blotting. The transcription of the cDNA into mRNA and translation into the desired cytochrome P-450 protein was detected by Northern and Western blotting. The enzymatic activity was determined by the cytochrome P-450IA1-dependent oxidation of benzo[a]pyrene and 7-ethoxycoumarin. After exposure to benzo[a]pyrene, the mutant frequency increased in XEM1 and XEM2 cells and was higher than in V79 cells in the presence of an exogenous activating system. The mutant frequency was even more increased when XEM1 and XEM2 cells were exposed to the proximate mutagen (trans)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene.
Institut für Toxikologie, Johannes Gutenberg-Universität Mainz, Federal Republic of Germany.
S
S
Dogra
J
J
Doehmer
H
H
Glatt
H
H
Mölders
P
P
Siegert
T
T
Friedberg
A
A
Seidel
F
F
Oesch
D000818:Animals; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D015139:Blotting, Southern; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004101:Dihydroxydihydrobenzopyrenes / Q000494:pharmacology; D015870:Gene Expression; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D015345:Oligonucleotide Probes; D010957:Plasmids; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D014162:Transfection
D004101:Dihydroxydihydrobenzopyrenes; D015345:Oligonucleotide Probes; C017228:benzo(a)pyrene 7,8-dihydrodiol; D001564:Benzo(a)pyrene; D012313:RNA; D004247:DNA; D003577:Cytochrome P-450 Enzyme System