Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression.
The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol.min-1.mg microsomal protein-1 vs 68 +/- 48 nmol.min-1.mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol.min-1.mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.
Mapoles
J
J
HepatoBiliary Research Center, VAMC, University of Colorado, Denver.
Berthou
F
F
Alexander
A
A
Simon
F
F
Ménez
J F
JF
D020001:1-Butanol; D000082:Acetaminophen / Q000494:pharmacology; D000818:Animals; D000440:Butanols / Q000378:metabolism; D002753:Chlorzoxazone / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D004128:Dimethylnitrosamine / Q000378:metabolism; D004050:Ditiocarb / Q000494:pharmacology; D005818:Genetic Engineering; D006801:Humans; D006900:Hydroxylation; D008861:Microsomes / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D010105:Oxygenases / Q000096:biosynthesis; D016716:PC12 Cells; D014162:Transfection
D000440:Butanols; D000082:Acetaminophen; D020001:1-Butanol; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D010105:Oxygenases; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D002753:Chlorzoxazone; D004128:Dimethylnitrosamine