Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens.
We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.
Davies
R L
RL
Gentest Corporation, Woburn, MA 01801.
Crespi
C L
CL
Rudo
K
K
Turner
T R
TR
Langenbach
R
R
D001564:Benzo(a)pyrene / Q000378:metabolism / Q000494:pharmacology*; D001711:Biotransformation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004128:Dimethylnitrosamine / Q000494:pharmacology*; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015183:Restriction Mapping; D014162:Transfection
D001564:Benzo(a)pyrene; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D004851:Epoxide Hydrolases; D004128:Dimethylnitrosamine