Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression

Eur J Biochem. 1993 Jun 15;214(3):735-45. doi: 10.1111/j.1432-1033.1993.tb17975.x.


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 microsomal protein-1 vs 68 +/- 48 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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Butanol
  • Acetaminophen / pharmacology
  • Animals
  • Butanols / metabolism
  • Chlorzoxazone / metabolism
  • Cytochrome P-450 CYP2E1
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Cytochrome P-450 Enzyme System / drug effects
  • Cytochrome P-450 Enzyme System / genetics*
  • Dimethylnitrosamine / metabolism
  • Ditiocarb / pharmacology
  • Genetic Engineering
  • Humans
  • Hydroxylation
  • Microsomes / enzymology
  • Oxidoreductases, N-Demethylating / biosynthesis*
  • Oxidoreductases, N-Demethylating / drug effects
  • Oxidoreductases, N-Demethylating / genetics*
  • Oxygenases / biosynthesis
  • PC12 Cells
  • Transfection


  • Butanols
  • Acetaminophen
  • 1-Butanol
  • Cytochrome P-450 Enzyme System
  • Ditiocarb
  • Oxygenases
  • Cytochrome P-450 CYP2E1
  • Oxidoreductases, N-Demethylating
  • Chlorzoxazone
  • Dimethylnitrosamine