Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens

Abstract

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.

Identification of a Rat Oltipraz-inducible UDP-glucuronosyltransferase (UGT1A7) with Activity Towards Benzo(a)pyrene-7,8-dihydrodiol

“…To investigate its catalytic activity, we utilized the AHH-1 system (24). A potential disadvantage of this system is the instability of the Epstein-Barr virus-based episomal expression plasmid.…”

“…Expression of the LC14-encoded UGT-The LC14 cDNA was ligated into the SalI site of pMF6 (24). Colonies containing plasmid with the LC14 insert in the correct (pMF6-LC14-3) or reverse (pMF6-LC14-23) orientations were selected and amplified.…”

Genotyping for polymorphisms in xenobiotic metabolism as a predictor of disease susceptibility.

“…This enzyme may also be expressed in other tissues, including nasal tissue (19). CYP2A6 carries out 7-hydroxylation of coumarin but also appears to activate certain procarcinogens, including hexamethylphosphoramide (19), Nnitrosodimethylamine (20), NNK (14) and aflatoxin B1 (21). Using coumarin as a probe drug, the formation of 7-hydroxycoumarin has been investigated in a group of volunteers.…”

Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons associated with fossil fuels combustion.

“…These cells were derived from an L3 variant (30) (32). L3 cells have a 2-fold higher CYPlAI activity and a lower background mutation frequency at the tk locus than AHH-1 cells.…”